Pseudorabies virus protein

ABSTRACT

The present invention provides recombinant DNA molecules comprising a sequence encoding a pseudorabies virus (PRV) glycoprotein selected from the group consisting of gI, gp50, and gp63, host cells transformed by said recombinant DNA molecule sequences, the gI, gp50 and gp63 polypeptides. The present invention also provides subunit vaccines for PRV, methods for protecting animals against PRV infection and methods for distinguishing between infected and vaccinated animals.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part application ofSer. No. 07/100,817, filed 29 Jun. 1987, now pending.

FIELD OF INVENTION

[0002] This invention relates to DNA sequences encoding pseudorabiesvirus glycoproteins and polypeptides related thereto. These DNAsequences are useful for screening animals to determine whether they areinfected with PRV and also for expressing the glycoproteins encodedthereby.

BACKGROUND OF THE INVENTION

[0003] Pseudorabies virus (PRV) is a disease which infects many speciesof animals worldwide. PRV infections are variously called infectiousBulbar paralysis, Aujeszky's disease, and mad itch. Infections are knownin important domestic animals such as swine, cattle, dogs, cats, sheep,rats and mink. The host range is very broad and includes most mammalsand, experimentally at least, many kinds of birds (for a detailed listof hosts, see D. P. Gustafson, “Pseudorabies”, in Diseases of Swine, 5thed., A. D. Leman et al., eds., (1981)). For most infected animals thedisease is fatal. Adult swine and possibly rats, however, are not killedby the disease and are therefore carriers.

[0004] Populations of swine are particularly susceptible to PRV.Although the adult swine rarely show symptoms or die from the disease,piglets become acutely ill when infected and death usually ensues in 24to 48 hours often without specific clinical signs (T. C. Jones and R. D.Hunt, Veterinary Pathology, 5th ed., Lea & Febiger (1983)).

[0005] PRV vaccines have been produced by a variety of techniques andvaccination in endemic areas of Europe has been practiced for more than15 years. Losses have been reduced by vaccination, but vaccination hasmaintained the virus in the environment. No vaccine has been producedthat will prevent infection. Vaccinated animals that are exposed tovirulent virus survive the infection and then shed more virulent virus.Vaccinated animals may therefore harbor a latent infection that canflare up again. (See, D. P. Gustafson, supra).

[0006] Live attenuated and inactivated vaccines for PRV are availablecommerciallv in the United States and have been approved bv the USDA(See, C. E. Aronson, ed., Veterinary Pharmaceuticals & Biologicals.(1983)).

[0007] Because adult swine are carriers of PRV. manv staees haveinstituted screening programs to detect infeczed animals. DNA DNAhybridization can be used to diagnose actively infected animalsutilizing the DNA sequence of the instant invention. Some of the PRVglycoproteins of the present invention are also useful in producingdiagnostics for PRV infections and also to produce vaccines against PRV.

[0008] PRV is a herpesvirus. The herpesviruses generally are among themost complex of animal viruses. Their genomes encode at least 50 virusspecific proteins and contain upwards of 150,000 nucleotides. Among themost immunologically reactive proteins of herpesviruses are theglycoproteins found, among other places, in virion membranes and themembranes of infected cells. The literature on PRV glycoproteins refersto at least four viral glycoproteins (T. Ben-Porat and A. S. Kaplan,Virology, 41, pp. 265-73 (1970); A. S. Kaplan and T. Ben-Porat, Proc.Natl. Acad. Scm. USA, 66, pp. 799-806 (1970)).

INFORMATION DISCLOSURE

[0009] M. W. Wathen and L. K. Wathen, J. Virol., 51, pp. 57-62 (1984)refer to a PRV containing a mutation in a viral glycoprotein (gpSO) anda method for selecting the mutant utilizing neutralizing monoclonalantibody directed against gp50. Wathen and Wathen also indicate that amonoclonal antibody directed against gp50 is a strong neutralizer ofPRV, with or without the aid of complement, and that polyvalent immuneserum is highly reactive against gpSO, therefore concluding that gp50may be one of the important PRV immunogens. On the other hand, it hasbeen reported that monoclonal antibodies that react with the 98,000 MWenvelope glycoprotein neutralize PRV infectivity but that monoclonalantibodies directed against some of the other membrane glycoproteinshave very little neutralizing activity (H. Hampl, et al., J. Virol., 52,pp. 583-90 (1984); and T. Ben-Porat and A. S. Kaplan, “Molecular Biologyof Pseudorabies Virus”, in B. Roizman ed., The Herpesviruses, 3, pp.105-73 (1984)).

[0010] L. M. K. Wathen, et al., Virus Research, 4, pp. 19-29 (1985)refer to the production and characterization of monoclonal antibodiesdirected against PRV glycoproteins identified as gp50 and gp83 and theiruse for passively immunizing mice against PRV infection.

[0011] A. K. Robbins, et al. “Localization of a Pseudorabies VirusGlycoprotein Gene Using an E. coli Expression Plasmid Library”. inHerpesvirus. pp. 551-61 (b 1984). refer to the construction of a libraryof E. coli plasmids containing PRV DNA. They also refer to theidentification of a PRV gene that encodes glycoproteins of 74.000 and92,000 .MW. They do not refer to the glycoproteins of the instantinvention.

[0012] A. K. Robbins, et al., European patent application No. 85400704.4(publication No. 0 162 738) refers to the isolation, cloning andexpression of PRV glycoproteins identified as glI and glII. They do notrefer to the PRV glycoproteins of the instant invention.

[0013] T. C. Mettenleiter, et al., “Mapping of the Structural Gene ofPseudorabies Virus Glycoprotein A and Identification of TwoNon-Glycosylated Precursor Polypeptides”, J. Virol., 53, pp. 52-57(1985), refer to the mapping of the coding region of glycoprotein gA(which they equate with gI) to the BamHI 7 fragment of PRV DNA. Theyalso state that the BamHI 7 fragment codes for at least three otherviral proteins of 65K, 60K, and 40K MW. They do not disclose or suggestthe DNA sequence encoding the glycoproteins of the instant invention orthe production of such poly-peptides by recombinant DNA methods.

[0014] B. Lomniczi, et al., “Deletions in the Cenomes of PseudorabiesVirus Vaccine Strains and Existence of Four Isomers of the Genomes”, J.Virol., 49, pp. 970-79 (1984), refer to PRV vaccine strains that havedeletions in the unique short sequence between 0.855 and 0.882 mapunits. This is in the vicinity of the gI gene. T. C. Mettenleiter, etal., “Pseudorabies Virus Avirulent Strains Fail to Express a MajorGlycoprotein”, J. Virol., 56, pp. 307-11 (1985), demonstrated that threecommercial PRV vaccine strains lack glycoprotein gI. We have also foundrecently that the Bartha vaccine strain contains a deletion for most ofthe gp63 gene.

[0015] T. J. Rea et al., J. Virol., 54, pp. 21-29 (1985), refers to themapping and the sequencing of the gene for the PRV glycoprotein thataccumulates in the medium of infected cells (gX). Included among theflanking sequences of the gX gene shown therein is a small portion ofthe gp50 sequence, specifically beginning at base 1682 of FIG. 6therein. However, this sequence was not identified as the gp50 sequence.Furthermore, there are errors in the sequence published by Rea et al.Bases 1586 and 1603 should be deleted. Bases should be inserted betweenbases 1708 and 1709, bases 1737 and 1738. bases 1743 and 1744 and bases1753 and 1754. The consequence of these errors a,n the published partialsequence for gp50 is a frameshift. Translation of the open reading framebeginning at the AUG start site would give an incorrect amino acidsequence for the gp50 glycoprotein.

[0016] European published patent application 0 133 200 refers to adiagnostic antigenic factor to be used together with certainlectin-bound PRV glycoprotein subunit vaccines to distinguish carriersand noncarriers of RV.

SUMMARY OF INVENTION

[0017] The present invention provides recombinant DNA moleculescomprising DNA sequences encoding polypeptides displaying PRVglycoprotein antigenicity.

[0018] More particularly, the present invention provides host cellstransformed with recombinant DNA molecules comprising the DNA sequencesset forth in Charts A, B, and C, and fragments thereof.

[0019] The present invention also provides polypeptides expressed byhosts transformed with recombinant DNA molecules comprising DNAsequences of the formulas set forth in Charts A, B, and C, andimmunologically functional equivalents and immunogenic fragments andderivatives of the polypeptides.

[0020] More particularly, the present invention provides polypeptideshaving the formulas set forth in Charts A, B, and C, immunogenicfragments thereof and immunologically functional equivalents thereof.

[0021] The present invention also provides recombinant DNA moleculescomprising the DNA sequences encoding pseudorabies virus glycoproteinsgp50, gp63, gI or immunogenic fragments thereof operatively linked to anexpression control sequence.

[0022] The present invention also provides vaccines comprising gp50 andgp63 and methods of protecting animals from PRV infection by vaccinatingthem with these polypeptides.

DETAILED DESCRIPTION OF INVENTION

[0023] The existence and location of the gene encoding glycoprotein gp50of PRV was demonstrated by M. W. Wathen and L. M. Wathen, supra.

[0024] The glycoprotein encoded by the gene was defined as aglycoprotein that reacted with a particular monoclonal antibody. Thisglycoprotein did not correspond to any of the previously known PRVglycoproteins. Wathen and %;athen mapped a mutation resistant to themonoclonal antibody, which, based on precedent in herpes simplex virus(e.g., T. C. Holland et al., J. Virol.. 52. pp.566-74 (1984)). maps thelocation of the structural gene for gp50. Wathen and Wathen mapped thegp50 gene to the smaller SalI/BamHI fragment from within the BamHI 7fragment of PRV. Rea et al, supra, have mapped the PRV glycoprotein gXgene to the same region.

[0025] The PRV gp63 and gI genes were isolated by screening PRV DNAlibraries constructed in the bacteriophage expression vector Agtll (J.G. Timmins, et al., “A method for Efficient Gene Isolation from PhageAgtll Libraries: Use of Antisera to Denatured, Acetone-PrecipitatedProteins”, Gene. 39, pp. 89-93 (1985); R. A. Young and R. W. Davis,Proc. Natl.Acad. Sci. USA. 80. pp. 1194-98 (1983); R. A. Young and R. W.Davis, Science, 222, pp.778-82 (1983)).

[0026] PRV genomic DNA derived from PRV Rice strain originally obtainedfrom D. P. Gustafson at Purdue University was isolated from thecytiplasm of PRV-infected Vero cells (ATCC CCL 81). The genomic DNA wasfragmented by sonication and then cloned into Agtll to produce a A/PRVrecombinant (IPRV) DNA library.

[0027] Antisera for screening the XPRV library were produced byinoculating mice with proteins isolated from cells infected with PRV(infected cell proteins or ICP's) that had been segregated according tosize on SDS gels, and then isolating the antibodies. The APRV phages tobe screened were plated on a lawn of E. coli. Xgtll contains a uniquecloning site in the 3′ end of the lacZ gene. Foreign DNA's inserted inthis unique site in the proper orientation and reading frame produce, onexpression, polypeptides fused to β-galactosidase. A nitrocellulosefilter containing an inducer of lacZ transcription to enhance expressionof the PRV DNA was laid on top of the lawn. After the fusionpolypeptides expressed by APRV's had sufficient time to bind to thenitrocellulose filters, the filters were removed from the lawns andprobed with the mouse antisera. Plaques producing antigen that bound themice antisera were identified by probing with a labeled antibody for themouse antisera.

[0028] Plaques that gave a positive signal were used to transform an E.coli host (Y1090, available from the ATCC, Rockville, Md. 20852). Thecultures were then incubated overnight to produce the APRV phage stocks.These phage stocks were used to infect E. coli K95 (D. Friedman. in sheBacteriophage Lambda, pp. 733-38. A. D. Hershey, ed. (1931))Polypeptides produced bv the transformed E. coli K95 were purified bypreparative gel electrophoresis Polypeptides that were overproduced (dueto induction of transcription of the lacZ gene), having molecularweights greater than 116,000 daltons, and which were also absent fromAgtll control cultures were β-galactosidase-PRV fusion proteins. Eachindividual fusion protein was then injected into a different mouse toproduce antisera.

[0029] Labeled PRV ICP's were produced by infecting Vero cells growingin a medium containing, for example, ¹⁴C-glucosamine (T. J. Rea, et al.,supra.). The fusion protein antisera from above were used toimmunoprecipitate these labeled ICP's. The polypeptides so precipitatedwere analyzed by gel electrophoresis. One of them was a 110 kd MWglycoprotein (gI) and another a 63 kd MW glycoprotein (gp63). The genescloned in the phages that produced the hybrid proteins raising anti-gIand anti-gp63 serum were thus shown to be the gI and gp63 genes. Thesegenes were found to map within the BamHI 7 fragment of the PRV genome(T. J. Rea, et al., supra.) as does the gp50 sequence (see Chart D). ThegI location is in general agreement with the area where Mettenleiter, etal., supra, had mapped the gI gene. However, Mettenleiter, et al.implied that the gI gene extends into the BamHI 12 fragment which itdoes not.

[0030] This XPRV gene isolation method is rapid and efficient whencompared to DNA hybridization and to in vitro translation of selectedmRNAs. Because purified glycoproteins were unavailable, we could notconstruct, rapidly, oligonucleotide probes from amino acid sequencedata, nor could we raise highly specific polyclonal antisera. There-fore we used the method set forth above.

[0031] As mentioned above, the genes encoding gp50, gp63, and gI mappedto the BamHI 7 fragment of the PRV DNA. The BamHI 7 fragment from PRVcan be derived from plasmid pPRXhl (also known as pUC1129) and fragmentsconvenient for DNA sequence analysis can be derived by standardsubcloning procedures. Plasmid pUC1129 is available from E. coli HB101,NRRL B-15772. This culture is available from the per- manent collectionof the Northern Regional Research Center Fermentation Laboratory (NRRL),U.S. Department of Agriculture, in Peoria, Ill., U.S.A.

[0032]E. coli HB101 containing pUC1129 can be grown up in L-broth bvwell knowe procedures. Typically the culture is grove. to an opticaldensity of 0.6 after which chloramonp;enicol is added and the culture isleft to shake overnight. The culture is then lsed by. e.g., using highsalt SDS and the supernatant is subjected to a cesium chloride/ ethidiumbromide equilibrium density gradient centrifugation to yield theplasmids.

[0033] The availability of these gene sequences permits directmanipulation of the genes and gene sequences which allows modificationsof the regulation of expression and/or the structure of the proteinencoded by the gene or a fragment thereof. Knowledge of these genesequences also allows one to clone the corresponding gene, or fragmentthereof, from any strain of PRV using the known sequence as ahybridization probe, and to express the entire protein or fragmentthereof by recombinant techniques generally known in the art.

[0034] Knowledge of these gene sequences enabled us to deduce the aminoacid sequence of the corresponding polypeptides (Charts A-C). As aresult, fragments of these polypeptides having PRV immunogenicity can beproduced by standard methods of protein synthesis or recombinant DNAtechniques. As used herein, immunogenicity and antigenicity are usedinterchangeably to refer to the ability to stimulate any type ofadaptive immune response, i.e., antigen and antigenicity are not limitedin meaning to substances that stimulate the production of antibodies.

[0035] The primary structures (sequences) of the genes coding for gp50,gp63, and gI also are set forth in Charts A-C.

[0036] The genes or fragments thereof can be extracted from pUC1129 bydigesting the plasmid DNA from a culture of NRRL B-15772 withappropriate endonuclease restriction enzymes. For example, the BamHI 7fragment may be isolated by digestion of a preparation of pUC1129 withBamHI, and isolation by gel electrophoresis.

[0037] All restriction endonucleases referred to herein are commerciallyavailable and their use is well known in the art. Directions for usegenerally are provided by commercial suppliers of the restrictionenzymes.

[0038] The excised gene or fragments thereof can be ligated to variouscloning vehicles or vectors for use in transforming a host cell. Thevectors preferably contains DNA sequences to initiate, control andterminate transcription and translation (which together compriseexpression) of the PRV glycoprotein genes and are, therefore,operativelv linked thereto. These “expression control sequences” arepreferably compatible with the host cell to be transformed. Ven the hostcell is a higher animal cell, e.g., a mammalian cell, the naturallyoccurring expression control sequences of the glycoprotein genes can beemployed alone or together with heterologous expression controlsequences. Heterologous sequences may also be employed alone. Thevectors additionally preferably contain a marker gene (e.g., antibiotresistance) to provide a phenotypic trait for selection of transformedhost cells. Additionally a replicating vector will contain a replicon.

[0039] Typical vectors are plasmids, phages, and viruses that infectanimal cells. In essence, one can use any DNA sequence that is capableof transforming a host cell.

[0040] The term host cell as used herein means a cell capable of beingtransformed with the DNA sequence coding for a polypeptide displayingPRV glycoprotein antigenicity. Preferably, the host cell is capable ofexpressing the PRV polypeptide or fragments thereof. The host cell canbe procaryotic or eucaryotic. Illustrative procaryotic cells arebacteria such as E. coli, B. subtilis, Pseudomonas, and B.stearothermophilus. Illustrative eucaryotic cells are yeast or higheranimal cells such as cells of insect, plant or mammalian origin.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions may also be used. Mammalian celllines include, for example, VERO and HeLa cells, Chinese hamster ovary(CHO) cell lines, WI38, BHK, COS-7 or MDCK cell lines. Insect cell linesinclude the Sf9 line of Spodoptera frugiperda (ATCC CRL1711). A summaryof some available eucaryotic plasmids, host cells and methods foremploying them for cloning and expressing PRV glycoproteins can be foundin K. Esser, et al., Plasmids of Eukaryotes (Fundamentals andApplications), Springer-Verlag (1986) which is incorporated herein byreference.

[0041] As indicated above, the vector, e.g., a plasmid, which is used totransform the host cell preferably contains compatible expressioncontrol sequences for expression of the PRV glycoprotein gene orfragments thereof. The expression control sequences are, therefore,operatively linked to the gene or fragment. When the host cells arebacteria, illustrative useful expression control sequences include thetrp promoter and operator (Goeddel, et al., Nucl. Acids Res., 8, 4057(1980)); the lac promoter and operator (Chang, et al., Nature, 275, 615(1978)); the outer membrane protein promoter (EMBO J., 1, 771-775(1982)); the bacteriophage λ promoters and operators (Nucl. Acids Res.,11, 4677-4688 (1983)); the α-amylase (B. subtilis) promoter andoperator, termination sequences and other expression enhancement andcontrol sequences compatible with the selected host cell. When the hostcell is yeast, illustrative useful expression control sequences include,e.g., α-mating factor. For insect cells the polyhedrin promoter ofbaculoviruses can be used (Mol. Cell. Biol., 3, pp. 2156-65 (1983)).When the host cell is of insect or mammalian origin illustrative usefulexpression control sequences include, e.g., the SV-40 promoter (Science,222, 524-527 (1983)) or, e.g., the metallothionein promoter (Nature,296, 39-42 (1982)) or a heat shock promoter (Voellmy, et al., Proc.Natl. Acad. Sci. USA, 82, pp. 4949-53 (1985)). As noted above, when thehost cell is mammalian one may use the expression control sequences forthe PRV glycoprotein gene but preferably in combination withheterologous expression control sequences.

[0042] The plasmid or replicating or integrating DNA material containingthe expression control sequences is cleaved using restriction enzymes,adjusted in size as necessary or desirable, and ligated with the PRVglycoprotein gene or fragments thereof by means well known in the art.When yeast or higher animal host cells are employed, polyadenylation orterminator sequences from known yeast or mammalian genes may beincorporated into the vector. For example, the bovine growth hormonepolyadenylation sequence may be used as set forth in Europeanpublication number 0 093 619 and incorporated herein by reference.Additionally gene sequences to control replication of the host cell maybe incorporated into the vector.

[0043] The host cells are competent or rendered competent fortransformation by various means. When bacterial cells are the host cellsthey can be rendered competent by treatment with salts, typically acalcium salt, as generally described by Cohen, PNAS, 69, 2110 (1972). Ayeast host cell generally is rendered competent by removal of its cellwall or by other means such as ionic treatment (J. Bacteriol., 153,163-168 (1983)). There are several well-known methods of introducing DNAinto animal cells including, e.g., calcium phosphate precipitation,fusion of the recipient cells with bacterial protoplasts containing theDNA treatment of the recipient cells with liposomes containing the DNA,and microinjection of the DNA directly into the cells.

[0044] The transformed cells are grown up by means well known in the art(Molecular Cloning, Maniatis, T., et al., Cold Spring Harbor Laboratory,(1982); Biochemical Methods In Cell Culture And Virology, Kuchler, R.J., Dowden, Hutchinson and Ross, Inc., (1977); Methods In YeastGenetics, Sherman, F., et al., Cold Spring Harbor Laboratory, (1982))and the expressed PRV glycoprotein or fragment thereof is harvested fromthe cell medium in those systems where the protein is excreted from thehost cell, or from the cell suspension after disruption of the host cellsystem by, e.g., mechanical or enzymatic means which are well known inthe art.

[0045] As noted above, the amino acid sequences of the PRV glycoproteinsas deduced from the gene structures are set forth in Charts A-C.Polypeptides displaying PRV glycoprotein antigenicity include thesequences set forth in Chart A-C and any portions of the polypeptidesequences which are capable of eliciting an immune response in ananimal, e.g., a mammal, which has been injected with the polypeptidesequence and also immunogenically functional analogs of thepolypeptides.

[0046] As indicated hereinabove the entire gene coding for the PRVglycoprotein can be employed in constructing the vectors andtransforming the host cells to express the PRV glycoprotein, orfragments of the gene coding for the PRV glycoprotein can be employed,whereby the resulting host cell will express polypeptides displaying PRVantigenicity. Any fragment of the PRV glycoprotein gene can be employedwhich results in the expression of a polypeptide which is an immunogenicfragment of the PRV glycoprotein or an analog thereof. As is well knownin the art, the degeneracy of the genetic code permits easy substitutionof base pairs to produce functionally equivalent genes and fragmentsthereof encoding polypeptides displaying PRV glycoprotein antigenicity.These functional equivalents also are included within the scope of theinvention.

[0047] Charts D-S are set forth to illustrate the constructions of theExamples. Certain conventions are used to illustrate plasmids and DNAfragments as follows:

[0048] (1) The single line figures represent both circular and lineardouble-stranded DNA.

[0049] (2) Asterisks (*) indicate that the molecule represented iscircular. Lack of an asterisk indicates the molecule is linear.

[0050] (3) Endonuclease restriction sites of interest are indicatedabove the line.

[0051] (4) Genes are indicated below the line.

[0052] (5) Distances between genes and restriction sites are not toscale. The figures show the relative positions only unless indicatedotherwise.

[0053] Most of the recombinant DNA methods employed in practicing thepresent invention are standard procedures, well known to those skilledin the art, and described in detail, for example, in Molecular Cloning,T. Maniatis, et al., Cold Spring Harbor Laboratory, (1982) and B.Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons(1984), which are incorporated herein by reference.

EXAMPLE 1

[0054] In this example we set forth the sequencing, cloning andexpression of PRV glycoprotein gp50.

[0055] 1. Sequencing of the gp50 Gene

[0056] The BamHI 7 fragment of PRV Rice strain DNA (Chart D) whichencodes the gp50 gene is isolated from pPRXh1 [NRRL B-15772], supra.,and subcloned into the BamHI site of plasmid pBR322 (Maniatis et al.,supra.).

[0057] Referring now to Chart E, the fragment is further subcloned usingstandard procedures by digesting BamHI 7 with PvuII, isolating the twoBamHI/PvuII fragments (1.5 and 4.9 kb) and subcloning them between theBamHI and PvuII sites of pBR322 to produce plasmids pPR28-4 and pPR28-1incorporating the 1.5 and 4.9 kb fragments respectively (see also, Reaet al., supra.). These subclones are used as sources of DNA for DNAsequencing experiments.

[0058] Chart F shows various restriction enzyme cleavage sites locatedin the gp50 gene and flanking regions. The 1.5 and 4.9 kb fragmentssubcloned above are digested with these restriction enzymes. Each of theends generated by the restriction enzymes is labeled with γ-³²P-ATPusing polynucleotide kinase and sequenced according to the method ofMaxam and Gilbert, Methods Enzymol., 65, 499-560 (1980). The entire geneis sequenced at least twice on both strands. The DNA sequence for gp50is set forth in Chart A. This DNA may be employed to detect animalsactively infected with PRV. For example, one could take a nasal orthroat swab, and then do a DNA/DNA hybridization by standard methods todetect the presence of PRV.

[0059] 2. Expression of gp50

[0060] Referring now to Chart G, a NarI cleavage site is located 35 basepairs upstream from the gp50 gene initiation codon. The first step inexpression is insertion of the convenient BamHI cleavage site at thepoint of the NarI cleavage site. Plasmid pPR28-4 from above is digestedwith restriction endonuclease NarI to produce DNA fragment 3 comprisingthe N-terminus encoding end of the gp50 gene and a portion of the gXgene. BamHI linkers are added to fragment 3 and the fragment is digestedwith BamHI to delete the gX sequence thus producing fragment 4. TheBamHI ends are then ligated to produce plasmid pPR28-4 Nar2.

[0061] Referring now to Chart H, we show the assembly of the completegp50 gene. pPG28-4 Nar2 is digested with BamHI and PvuII to producefragment 5 (160 bp) comprising the N-terminal encoding portion of thegp50 gene. Plasmid pPR28-1 from above is also digested with PvuII andBamHI to produce a 4.9 kb fragment comprising the C-terminal encodingportion of the gp50 gene (fragment 6). Plasmid pPGX1 (constructed as setforth in U.S. patent application Ser. No. 760,130)), or, alternatively,plasmid pBR322, is digested with BamHI, treated with bacterial alkalinephosphatase (BAP) and then ligated with fragments 5 and 6 to produceplasmid pBGP50-23 comprising the complete gp50 gene.

[0062] Referring now to Chart I, we show the production of plasmid pD50.Plasmid pBG50-23 is cut with restriction enzyme MaeIII (K. Schmid etal., Nucl. Acids Res., 12, p. 2619 (1984)) to yield a mixture offragments. The MaeIII ends are made blunt with T4 DNA polymerase andEcoRI linkers are added to the blunt ends followed by EcoRI digestion.The resulting fragments are cut with BamHI and a 1.3 kb BamHI/EcoRIfragment containing the gp50 gene (fragment 7) is isolated. PlasmidpSV2dhfr (obtained from the American Type Culture Collection, BethesdaResearch Laboratories, or synthesized according to the method of S.Subramani, et al., Mol. Cell. Biol., 2, pp. 854-64 (1981)) is digestedwith BamHI and EcoRI and the larger (5.0 kb) fragment is isolated toproduce fragment 8 containing the dihydrofolate reductase (dhfr) marker.Fragments 7 and 8 are then ligated to produce plasmid pD50 comprisingthe Ep50 gene and the dhfr marker

[0063] Referring now to Chart J, the immediate early promoter from humancytomegalovirus Towne strain is added upstream from the gp50 gene. pD50is digested with BamHI and treated with bacterial alkaline phosphataseto produce fragment 9. A 760 bp Sau3A fragment containing the humancytomegalovirus (Towne) immediate early promoter is isolated accordingto the procedure set forth in U.S. patent application Ser. No. 758,517to produce fragment 10 (see also, D. R. Thomsen, et al., Proc. Natl.Acad. Sci. USA, 81, pp. 659-63 (1984)). These fragments are then ligatedby a BamHI/Sau3A fusion to produce plasmid pDIE50. To confirm that thepromoter is in the proper orientation to transcribe the gp50 gene theplasmid is digested with SacI and PvuII and a 185 bp fragment isproduced.

[0064] Referring now to Chart K, the 0.6 kb PvuII/EcoRI fragmentcontaining the bovine growth hormone polyadenylation signal is isolatedfrom plasmid pGH2R2 (R. P. Woychik, et al., Nucl. Acids Res., 10, pp.7197-7210 (1982) by digestion with PvuII and EcoRI or from pSVCOW7(supra.) to produce fragment 11.

[0065] Fragment 11 is cloned between the EcoRI and SmaI cleavage sitesof pUC9 (obtained from Pharmacia/PL or ATCC) to give pCOWT1. pCOWT1 iscut with SalI, the ends made blunt with T4 DNA polymerase, EcoRI linkersare added, the DNA is cut with EcoRI, and the 0.6 kb fragment (fragment12) is isolated. This is the same as fragment 11 except that it has twoEcoRI ends and a polylinker sequence at one end.

[0066] Plasmid pDIE50 is cut with EcoRI, and fragment 12 is cloned intoit to produce plasmid pDIE50PA. Digestion with BamHI and PvuII producesa fragment of 1.1 kb in the case where the polyadenylation signal is inthe proper orientation. The plasmid can also be constructed by cloningin the polyadenylation sequence before the promoter.

[0067] Plasmid pDIE50PA is used to transfect CHO dhfr⁻ cells (DXB-11, G.Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci. USA, 77, pp. 4216-20(1980)) by calcium phosphate co-precipitation with salmon sperm carrierDNA (F. L. Graham and A. J. Van Der Eb, Virol., 52, pp. 456-67 (1973)).The dihydrofolate reductase positive (dhfr⁺) transfected cells areselected in Dulbecco's modified Eagle's medium plus Eagle'snon-essential amino acids plus 10% fetal calf serum. Selected dhfr⁺ CHOcells produce gp50 as detected by immunofluorescence with anti-gp50monoclonal antibody 3A-4, or by labelling with ¹⁴C-glucosamine andimmunoprecipitation with 3A-4. Monoclonal antibody 3A-4 is produced asdescribed in copending U.S. patent application Ser. No. 817,429, filedJan. 9, 1985. Immunoprecipitation reactions are performed as describedpreviously (T. J. Rea, et al., supra.) except for the following: Theextracts are first incubated with normal mouse serum, followed by washedStaphylococcus aureus cells, and centrifuged for 30 minutes in a BeckmanSW50.1 rotor at 40,000 rpm. After extracts are incubated with monoclonalor polyclonal antiserum plus S. Aureus cells, the cells are washed threetimes in 10 mM Tris HCl, pH 7.0, 1 mM EDTA, 0.1 M NaCl, 1% NP40 and 0.5%deoxycholate. Analysis of proteins is done on 11% SDS polyacrylamidegels (L. Morse, et al., J. Virol., 26, pp. 389-410 (1984)). Inpreliminary immunofluorescence assays it was found that 3A-4 reactedwith the pDIE50PA-transfected CHO cells but not with untransfected CHOcells. When the transfected CHO cells were labelled with¹⁴C-glucosamine, 3A-4 immunoprecipitated a labelled protein from cellscontaining pDIE50PA but not from control cells making human renin. Theprecipitated protein co-migrated on SDS-polyacrylamide gels with theprotein precipitated by 3A-4 from PRV-infected cells.

[0068] A clone of these transfected CHO cells producing gp50 can begrown in roller bottles, harvested in phosphate buffered saline plus 1mM EDTA, and mixed with complete Freund's adjuvant for use as a vaccine.

[0069] The gp50 gene can also be expressed in a vaccinia vector. In thisembodiment, after pBG50-23 is digested with MaeIII and the ends madeblunt with T4 DNA polymerase, the DNA is digested with BamHI. The 1.3BamHI/blunt-ended fragment containing the gp50 gene is isolated. PlasmidpGS20 (Mackett, et al., J. Virol., 49, pp. 857-64 (1984)) is cut withBamHI and SmaI, and the larger 6.5 kb fragment is isolated by gelelectrophoresis. These two fragments are ligated together to producepVV50. Plasmid pVV50 is transfected into CV-1 cells (ATCC CCL 70)infected with the WR strain of vaccinia virus (ATCC VR-119), andselected for thymidine kinase negative recombinants by plating on 143cells (ATCC CRL 8303) in 5-bromodeoxyuridine (BUdR) by the methodsdescribed by Mackett, et al. in DNA Cloning, Volume II: A PracticalApproach, D. M. Glover, ed., IRL Press, Oxford (1985). The resultingvirus, vaccinia-gp50, expressed gP50 in infected cells, as assayed bylabelling of the proteins of the infected cell with ¹⁴C-glucosamine andimmunoprecipitation with monoclonal antibody 3A-4.

EXAMPLE 2

[0070] In this example we set forth the protection of mice and swinefrom PRV challenge using the gp50 of Example 1 as an immunogenic agent.

[0071] In Tables 1-3, infra, the microneutralization assay was done asfollows: Serial two-fold dilutions of serum samples were done inmicrotiter plates (Costar) using basal medium Eagle (BME) supplementedwith 3% fetal calf serum and antibiotics. About 1000 pfu (50 μl) of PRVwere added to 50 μl of each dilution. Rabbit complement was included inthe virus aliquot at a dilution of 1:5 for the mouse serum assays butnot the pig serum assays. The samples were incubated for either 1 hr(swine sera) or 3 hrs (mouse sera) at 37^(•) C. After the incubationperiod, an aliquot (50 μl) of porcine kidney-15 (PK-15) cells (300,000cells/ml) in Eagle's Minimum Essential Medium was added to each serumper PRV sample. The samples were subsequently incubated at 37^(•) C. for2 days. Neutralizing titers represent the reciprocals of the highestdilutions which protected 50% of the cells from cytopathic effects.

[0072] Table 1 sets forth the protection of mice from challenge byvirulent PRV by immunization with gp50 produced in vaccinia virus. Micewere immunized by tail scarification with 25 μl or by the footpad routewith 50 μl. Mice were immunized 28 days prior to challenge (except micegiven PR-Vac which were immunized 14 days prior to challenge). TABLE 1Immunizing Dose Neutralizing % Agent (PFU) Route Titers^(a) Survival^(b)gp50 3.0 × 10⁷ Tail 1024 93 gp50 6.0 × 10⁷ Footpad 1024 100 gp50 7.5 ×10⁶ Tail 512 93 vaccinia^(c) 7.5 × 10⁶ Tail <8 27 BME^(d) — Tail <8 20PR-Vac^(e) — Footpad 512 90

[0073] Table 2 sets forth the protection of mice from challenge byvirulent PRV by immunization with gp50 produced in CHO cells. Mice wereimmunized at 28 days, 18 days and 7 days prior to challenge. Micereceived preparations with adjuvants subcutaneously on the first doseand preparations in saline intraperitoneally on the second and thirddoses. Each mouse received 10⁶ disrupted cells/dose. TABLE 2 ImmunizingNeutralizing Agent/Adjuvant Titers^(a) % Survival^(b) gp50/CFA^(c) 512100 (10/10) gp50/CFA (2 doses) ND  80 (4/5) gp50/IFA^(d) 1024  90 (9/10)gp50/saline 256 100 (3/3) CHO-renin^(e)/CFA <8  10 (1/10) Nontreated <8 0 (0/10) PR-Vac^(f) 4096  90 (9/10)

[0074] Table 3 sets forth the protection of swine from challenge byvirulent PRV by immunization with gp50 produced in CHO cells. Swine wereimmunized at 21 days and 7 days prior to challenge. Swine received 2×10⁷disrupted cells per dose. The first dose was mixed with completeFreund's adjuvant while the second dose was suspended in saline. Bothdoses were given intramuscularly. TABLE 3 Immunizing Geometric Mean %Agent/Adjuvant Titer^(a) Survival^(b) gp50/CFA 25 100 CHO-renin/CFA <8 0

[0075] These three tables demonstrate that gp50 can raise neutralizingantibodies and protect mice and swine from lethal PRV challenge.

[0076] In another aspect of the instant invention we produced aderivative of glycoprotein gp50 by removing the DNA coding for theC-terminal end of gp50. The resulting polypeptide has a deletion for theamino acid sequence necessary to anchor gp50 into the cell membrane.When expressed in mammalian cells this gp50 derivative is secreted intothe medium. Purification of this gp50 derivative from the medium for useas a subunit vaccine is much simpler than fractionation of whole cells.Removal of the anchor sequence to convert a membrane protein into asecreted protein was first demonstrated for the influenza hemagglutiningene (M. -J. Gething and J. Sambrook, Nature, 300, pp. 598-603 (1982)).

[0077] Referring now to Chart L, plasmid pDIE50 from above is digestedwith SalI and EcoRI. The 5.0 and 0.7 kb fragments are isolated. The 0.7kb fragment encoding a portion of gp50 is digested with Sau3A and a 0.5kb SalI/Sau3A fragment is isolated. To introduce a stop codon after thetruncated gp50 gene, the following oligonucleotides are synthesized:

[0078] 5′ GATCGTCGGCTAGTGAGTAGGTAGG 3′

[0079] 3′ CAGCCGATCACTCATCCATCCTTAA 5′

[0080] The 5.0 kb EcoRI/SalI fragment, the 0.5 kb SalI/Sau3A fragmentand the annealed oligonucleotides are ligated to produce plasmidpDIE50T. Digestion with EcoRI and SalI produces a 580 bp fragment.pDIE50T is cut with EcoRI and the 0.6 kb EcoRI fragment containing thebGH polyA site (fragment 12) is cloned in to produce plasmid pDIE50TPA.Digestion of pDIE50TPA with BamHI and PvuII yields a 970 bp fragmentwhen the polyadenylation signal is in the proper orientation.

[0081] pDIE50TPA is used to transfect CHO dhfr⁻ cells. Selected dhfr⁺CHO cells produce a truncated form of gp50 which is secreted into themedium as detected by labelling with ³⁵S-methionine andimmunoprecipitation.

EXAMPLE 3

[0082] In this example we set forth the isolation, cloning andsequencing of the gp63 and gI genes.

[0083] 1. Library Construction

[0084] PRV genomic DNA was prepared as described previously (T. J. Rea,et al., supra.). Fragments of 0.5-3.0 kb were obtained by sonicating thePRV genomic DNA of the PRV Rice strain twice for 4 sec each time atsetting 2 with a Branson 200 sonicator. After blunt ending the fragmentswith T4 DNA polymerase, the fragments were ligated to kinased EcoRIlinkers (T. Maniatis, et al., supra). After over-digestion with EcoRI(since PRV DNA does not contain an EcoRI site, methylation wasunnecessary), excess linkers were removed by agarose gelelectrophoresis. The PRV DNA fragments in the desired size range wereeluted by the glass slurry method, (B. Vogelstein and D. Gillespie,Proc. Natl. Acad. Sci. USA, 76, pp. 615-19 (1979)). A library of 61,000λ/PRV recombinants (λPRVs) was constructed by ligating 500 ng of PRV DNAfragments to 750 ng of EcoRI digested λgt11 (R. A. Young and R. W.Davis, supra.) DNA in 50 mM Tris (pH 7.4), 10 mM MgCl₂, 10 mMdithiothreitol, 1 mM spermidine, 1 mM ATP, 400 units of T4 DNA ligase(New England Biolabs), in a final volume of 10 μl. The ligated DNA waspackaged into bacteriophage λ virions using the Packagene extract(Promega Biotec, Madison, Wis.).

[0085] 2. λPRV Library Screening

[0086] The λPRV library was screened as previously described (J. G.Timmins, et al., supra.; R. A. Young and R. W. Davis, supra.). 20,000phages were screened per 150 mm LB-ampicillin plate. The screeningantisera were raised by injecting mice with size fractions of PRVinfected cell proteins (ICP's) eluted from SDS-polyacrylamide gels (J.G. Timmins, et al., supra.). Plaques giving positive signals uponscreening with antisera were picked from the agar plates with a sterilepasteur pipette, resuspended in 1 ml SM buffer (T. Maniatis, et al.,supra) and rescreened. The screening was repeated until the plaques werehomogeneous in reacting positively.

[0087] Approximately 43,000 λPRV recombinants were screened with mouseantisera to PRV infected Vero cell proteins, isolated fromSDS-polyacrylamide gels. Sixty positive λPRV phages were isolated.

[0088] 3. Phage Stock Preparation

[0089] High titer phage stocks (10¹⁰-10¹¹ pfu/ml) were prepared by theplate lysate method (T. Maniatis et al., supra). A single, well-isolatedpositive signal plaque was picked and resuspended in 1 ml SM. 100 μl ofthe suspension was adsorbed to 300 μl of E. coli Y1090 (available fromthe American Type Culture Collection (ATCC), Rockville, Md.) at 37^(•)C. for 15 min, diluted with 10 ml LB-top agarose, poured evenly on a 150mm LB-ampicillin plate and incubated overnight at 42^(•) C. The topagarose was gently scraped off with a flamed glass slide and transferredto a 30 ml Corex tube. 8 ml of SM and 250 μl of chloroform were added,mixed and incubated at 37^(•) C. for 15 min. The lysate was clarified bycentrifugation at 10,000 rpm for 30 min in the HB-4 rotor. The phagestock was stored at 4^(•) C. with 0.3% chloroform.

[0090] 4. Fusion Protein Preparation and Analysis

[0091] LB medium (Maniatis, et al., supra.) was inoculated 1:50 with afresh overnight culture of E. coli K95 (sup⁻, λ⁻, gal⁻, str^(r), nusA⁻;D. Friedman, supra.) and grown to an OD₅₅₀-0.5 at 30^(•) C. 25 ml ofculture was infected with λPRV phage at a multiplicity of 5 andincubated in a 42^(•) C. shaking water bath for 25 min, followed bytransfer to 37^(•) C. for 2-3 hours. The cells were pelleted at 5,000rpm for 10 min in the HB-4 rotor and resuspended in 100 μl of 100 mMTris (pH 7.8), 300 mM NaCl. An equal volume of 2×SDS-PAGE sample bufferwas added, and the sample was boiled for 10 min. 5 μl of each sample wasanalyzed by electrophoresis on analytical SDS-polyacrylamide gels asdescribed in L. Morse et al., J. Virol, 26, pp. 389-410 (1978). Thefusion polypeptide preparations were scaled up 10-fold for mouseinjections. The β-galactosidase/PRV fusion polypeptides were isolatedafter staining a strip of the gel with coomassie blue (L. Morse et al.,supra; K. Weber and M. Osborn, in The Proteins, 1, pp. 179-223 (1975)).Fusion polypeptide quantities were estimated by analytical SDS-PAGE.Cell lysates from λPRV infected E. coli K95 cultures wereelectrophoresed in 9.25% SDS-polyacrylamide gels. Overproducedpolypeptide bands with molecular weights greater than 116,000 daltons,absent from λgtll-infected controls, were β-galactosidase-PRV fusionpolypeptides. The β-galactosidase-PRV fusion polypeptides ranged in sizefrom 129,000 to 158,000 daltons. Approximately 50-75 μg of fusionpolypeptide was resuspended in complete Freund's adjuvant and injectedsubcutaneously and interperitoneally per mouse. Later injections weredone intraperitoneally in incomplete Freund's adjuvant.

[0092] 5. Antisera Analysis

[0093] Immunoprecipitations of ¹⁴C-glucosamine ICP's, ³⁵S-methionineICP's and ¹⁴C-glucosamine gX were done as previously described (T. J.Rea, et al., supra.). These techniques showed that gp63 and gI had beenisolated in a λgtll recombinant phage. We called these phages λ37 andλ36 (gp63) and λ23 (gI).

[0094] 6. λDNA Mini-preps

[0095] Bacteriophage were rapidly isolated from plate lysates (T. J.-Silhavy et al., Experiments With Gene Fusions, (1984)). 5% and 40%glycerol steps (3 ml each in SM buffer) were layered in an SW41 tube. Aplate lysate (−6 ml) was layered and centrifuged at 35,000 rpm for 60min at 4^(•) C. The supernatant was discarded and the phage pellet wasresuspended in 1 ml SM. DNAse I and RNAse A were added to finalconcentrations of 1 μg/ml and 10 μg/ml. After incubation at 37^(•) C.for 30 min, 200 μl of SDS Mix (0.25 M EDTA, 0.5 M Tris (pH 7.8), 2.5%SDS) and proteinase K (to 1 mg/ml) were added and incubated at 68^(•) C.for 30 min. The λDNA was extracted with phenol three times, extractedwith chloroform, and ethanol precipitated. An average 150 mm platelysate yields 5-10 μg of λDNA.

[0096] 7. λPRV DNA Analysis

[0097] PRV DNA was digested to completion with BamHI and KpnI,electrophoresed in 0.8% agarose and transferred to nitrocellulose by themethod of Southern (J. Mol. Biol., 98, pp. 503-17 (1975)). The blotswere sliced into 4 mm strips and stored desiccated at 20-25^(•). λPRVDNAs were nick-translated (Amersham) to specific activities ofapproximately 10⁸ cpm/μg. Pre-hybridization was done in 6×SSC, 30%formamide, 1×Denhardt's reagent (0.02% each of ficoll,polyvinylpyrrolidone and bovine serum albumin). 0.1% SDS, 50 μg/mlheterologous DNA at 70^(•) C. for 1 hour. Hybridization was done in thesame solution at 70^(•) C. for 16 hours. Fifteen minute washes were donetwice in 2×SSC, 0.1% SDS and twice in 0.1×SSC, and 0.1% SDS, all at20-25^(•). The blots were autoradiographed with an intensifying screenat −70^(•) C. overnight.

[0098] By Southern blotting the PRV glycoprotein genes contained in λ23,λ36 and λ37 mapped to the BamHI 7 fragment in the unique small region(see T. J. Rea, et al., supra.). Finer mapping of this fragment showedthat λ23 (gI) gene mapped distal to the gX gene and that λ37 mapped tothe internal region of BamHI 7, as shown in Chart D.

[0099] 8. Sequencing The gp63 and gI Genes

[0100] The PRV DNA in λ36 and λ37 was determined to contain a StuIcleavage site. There is only one StuI cleavage site in the BamHI 7fragment; therefore, the open reading frame that included the StuIcleavage site was sequenced. Chart E shows various restriction enzymecleavage sites located in the gp63 gene and flanking regions. BamHI 7was subcloned and digested with these restriction enzymes. Each of theends generated by the restriction enzymes was labeled with γ-³²P-ATPusing polynucleotide kinase and sequenced according to the method ofMaxam and Gilbert, Methods Enzymol., 65, 499-560 (1980).

[0101] Plasmid pPR28 is produced by cloning the BamHI 7 fragmentisolated from pUC1129 into plasmid pSV2 gpt (R. C. Mulligan and P. Berg,Proc. Natl. Acad. Sci. USA, 78, pp. 2072-76 (1981)).

[0102] Plasmid pPR28-1 was produced by digesting pPR28 with PvuII andthen recircularizing the piece containing the E. coli origin ofreplication and bla gene to produce a plasmid comprising a 4.9 kbPvuII/BamHI 7 PRV fragment containing the DNA sequence for gI.

[0103] Chart N shows various restriction enzyme cleavage sites locatedin the gI gene and flanking regions. BamHI 7 was subcloned, digested,labeled and sequenced as set forth above.

[0104] The DNA sequences for glycoproteins gp63 and gI are set forth inCharts B and C respectively. This DNA may be employed to detect animalsactively infected with PRV. For example, one could take a nasal orthroat swab, and then by standard DNA/DNA hybridization methods detectthe presence of PRV.

EXAMPLE 4

[0105] In this example we set forth the expression of gI in mammaliancells.

[0106] A BamHI 7 fragment containing the gI gene is isolated fromplasmid pPR28 (see above) by digesting the plasmid with BamHI,separating the fragments on agarose gel and then excising the fragmentfrom the gel.

[0107] Referring now to Chart O, the BamHI 7 fragment isolated above isthen cloned into plasmid pUC19 (purchased from Pharmacia/PL) to produceplasmid A. Plasmid A is digested with DraI. DraI cleaves the pUC19sequence in several places, but only once in the BamHI 7 sequencebetween the gp63 and gI genes (Chart D) to produce, inter alia,fragment 1. BamHI linkers are ligated onto the DraI ends of thefragments, including fragment 1, and the resulting fragment mixture isdigested with BamHI. The product fragments are separated by agarose gelelectrophoresis and fragment 2 (2.5 kb) containing the gI gene ispurified. Fragment 2 is cloned into pUC19 digested with BamHI to produceplasmid pUCD/B. Of the two plasmids so produced, the plasmid containingthe gI gene in the proper orientation is determined by digesting theplasmids with BsmI and EcoRI; the plasmid in the proper orientationcontains a characteristic 750 bp BsmI/EcoRI fragment.

[0108] Referring now to Chart P, plasmid pUCD/B (Chart O) is digestedwith BsmI and EcoRI and the larger fragment (fragment 3, 4.4 kb) ispurified by agarose gel electrophoresis. The following twooligonucleotides are synthesized chemically by well-known techniques orare purchased from a commercial custom synthesis service:

[0109] 5′ CGCCCCGCTTAAATACCGGGAGAAG 3′

[0110] 5′ AATTCTTCTCCCGGTATTTAAGCGGGGCGGG 3′

[0111] These oligonucleotides are ligated to fragment 3 to replace thecoding sequence for the C-terminus of the gI gene which was deleted bythe BsmI cleavage. The resulting plasmid, pGI, contains a completecoding region of the gI gene with a BamHI cleavage site upstream and anEcoRI cleavage site downstream from the gI coding sequences.

[0112] Plasmid pGI is digested with EcoRI and BamHI and a 1.8 kbfragment comprising the gI gene (fragment 4) is purified on an agarosegel.

[0113] Plasmid pSV2dhfr, (supra.) is cut with EcoRI, and is then cutwith BamHI to produce fragment 5 (5.0 kb) containing the dhfr marker,which is isolated by agarose gel electrophoresis. Then fragments 4, and5 are ligated to produce plasmid pDGI which comprises the dihydrofolatereductase and ampicillin resistance markers, the SV40 promoter andorigin of replication, and the gI gene.

[0114] Referring now to Chart Q, the immediate early promoter from humancytomegalovirus Towne strain is added upstream from the gI gene. PlasmidpDGI is digested with BamHI to produce fragment 6. The humancytomegalovirus (Towne) immediate early promoter is isolated (supra.) toproduce fragment 7. Fragments 6 and 7 are then ligated to produceplasmid pDIEGIdhfr. To confirm that the promoter is in the properorientation the plasmid is digested with SacI and BstEII restrictionenzymes. The production of an about 400 bp fragment indicates properorientation.

[0115] A 0.6 kb PvuII/EcoRI fragment containing the bovine growthhormone polyadenylation signal is isolated from the plasmid pSVCOW7(supra.) to produce fragment 8. Fragment 8 is cloned across theSmaI/EcoRI sites of pUC9 (supra.) to produce plasmid pCOWT1. pCOWT1 iscut with SalI, treated with T4 DNA polymerase, and EcoRI linkers areligated on. The fragment mixture so produced is then digested with EcoRIand a 0.6 kb fragment is isolated (fragment 9). Fragment 9 is clonedinto the EcoRI site of pUC19 to produce plasmid pCOWT1E. pCOWT1E isdigested with EcoRI to produce fragment 10 (600 bp).

[0116] Plasmid pDIEGIdhfr is digested with EcoRI and ligated withfragment 10 containing the bGH polyadenylation signal to produce plasmidpDIEGIPA. The plasmid having the gI gene in the proper orientation isdemonstrated by the production of a 1400 bp fragment upon digestion withBamHI and BstEII.

[0117] The resulting plasmid is transfected into dhfr⁻ Chinese hamsterovary cells and dhfr⁺ cells are selected to obtain cell lines expressinggI (Subramani, et al, Mol. Cell Biol., 1, pp.854-64 (1981)). Theexpression of gI is amplified by selecting clones of transfected cellsthat survive growth in progressively higher concentrations ofmethotrexate (McCormick, et al, Mol. Cell Biol., 4, pp. 166-72 (1984).

EXAMPLE 5

[0118] In this example we set forth the expression of gp63 in mammaliancells.

[0119] The BamHI 7 fragment of PRV DNA (supra.) is isolated from pPRXh1[NRRL B-15772], and subcloned into the BamHI site of plasmid pBR322 asin Example 1 for use in sequencing and producing more copies of the gp⁶³gene.

[0120] Referring now to Chart R, from within BamHI 7 a 1.9 kbBstEII/K-pnI fragment (fragment 1) is subcloned by cutting BamHI 7 withBstEII, treating the ends with T4 DNA polymerase, and then cutting withKpnI. Fragment 1 is isolated and cloned between the KpnI and SmaI sitesin pUC19 (purchased from Pharmacia/PL, Piscataway, N.J.) to yieldplasmid pPR28-1BK.

[0121] Plasmid pPR28-1BK is cut with DraI plus MaeIII to yield fragment2 (1.1 kb). The DraI cleavage site is outside the coding region of thegp63 gene and downstream from its polyadenylation signal. The MaeIIIcleavage site cuts 21 bases downstream from the ATG initiation codon ofthe gp63 gene. To replace the coding region removed from the gp63 gene,the following two oligonucleotides are synthesized chemically orpurchased from commercial custom synthesis services (fragment 4):

[0122] 5′ GATCCGCAGTACCGGCGTCGATGATGATGGTGGCGCGCGAC 3′

[0123] 3′ GCGTCATGGCCGCAGCTACTACTACCACCGCGCGCTGCACTG 5′

[0124] Plasmid pSV2dhfr, supra., is cut with EcoRI, treated with T4 DNApolymerase, then cut with BamHI and the larger (5.0 kb) fragment isisolated to produce fragment 4 containing the dhfr marker. Thenfragments 2, 3, and 4 are ligated to produce plasmid pGP63dhfr.

[0125] Referring now to Chart S, the immediate early promoter from humancytomegalovirus Towne strain is added upstream from the gp63 gene.pGP63dhfr is digested with BamHI and treated with bacterial alkalinephosphatase to produce fragment 5. A 760 bp Sau3A fragment containinghuman cytomegalovirus (Towne) immediate early promoter is isolated toproduce fragment 6. These fragments are then ligated to produce plasmidpIEGP63dhfr. To confirm that the promoter is in the proper orientationthe plasmid is digested with SacI and PvuII and a 150 bp fragment isproduced.

[0126] The resulting plasmid is transfected into dhfr⁻ Chinese hamsterovary cells and dhfr⁺ cells are selected to obtain cell lines expressinggp63. Since the levels of synthesis of gp63 by this system were too lowto detect by the methods we used, we produced the polypeptide invaccinia virus as set forth below.

EXAMPLE 6

[0127] In this example we set forth the expression of gp63 in vacciniavirus. The method used herein incorporates aspects of other synthesesreferred to above.

[0128] Fragments 1, 2, 3, and 4 are produced according to Example 5.

[0129] Plasmid pGS20 (Mackett, et al., J. Virol., 49, pp. 857-64 (1984))is cut with BamHI and SmaI, and the larger 6.5 kb fragment is isolatedby gel electrophoresis. Fragment 2, the oligonucleotides, and the pGS20fragment are ligated together to produce plasmid pVV63. This plasmid istransfected into CV-1 cells (ATCC CCL 70) infected with the WR strain ofvaccinia virus (ATCC VR-119), selected for thymidine kinase negativerecombinants by plating on 143 cells (ATCC CRL 8303) in BUdR by themethods described by Mackett, et al. in DNA Cloning, Volume II: APractical Approach, D. M. Glover, ed., IRL Press, Oxford (1985). Theresulting virus, vaccinia-gp63, expresses gp50 in infected cells, asassayed by labelling of the proteins of the infected cell with¹⁴C-glucosamine and immunoprecipitation with anti-gp63 antiserum.

[0130] The BamHI/EcoRI fragment from plasmid pGI, the DraI/MaeIIIfragment from plasmid pPR28-1BK, or the BamHI/MaeIII fragment frompBGP50-23 all described above, may also be treated with Bal31 andinserted in pTRZ4 (produced as set forth in copending U.S. patentapplication Ser. No. 606,307) as described in Rea, et al., supra., andused to transform E. coli. By this method, gp50, gp63, and gI can beproduced as a fusion protein in E. coli.

[0131] Also, by substituting, for example, pSV2neo (available from theAmerican Type Culture Collection) for pSV2dhfr in the above example, therecombinant plasmid comprising the PRV glycoprotein gene could betransformed into other host cells. Transformed cells would be selectedby resistance to antibiotic G418 which is encoded by the plasmid.

[0132] One can also express the polypeptides of the instant invention ininsect cells as follows: By putting a BamHI linker on the EcoRI site ofpD50 and digestion with BamHI, or putting a BamHI linker on the EcoRIsite of pGP63dhfr and digestion with BamHI, or by digestion of pUCD/Bwith BamHI, one obtains BamHI fragments containing the gp50, gp63, or gIgenes respectively. These BamHI fragments can be cloned into a BamHIsite downstream from a polyhedrin promoter in pAC373 (Mol. Cell. Biol.,5, pp. 2860-65 (1985)). The plasmids so produced can be co-transfectedwith DNA from baculovirus Autographa californica into Sf9 cells, andrecombinant viruses isolated by methods set forth in the article. Theserecombinant viruses produce gp50, gp63, or gI upon infecting Sf9 cells.

[0133] A vaccine prepared utilizing a glycoprotein of the instantinvention or an immunogenic fragment thereof can consist of fixed hostcells, a host cell extract, or a partially or completely purified PRVglycoprotein preparation from the host cells or produced by chemicalsynthesis. The PRV glycoprotein immunogen prepared in accordance withthe present invention is preferably free of PRV virus. Thus, the vaccineimmunogen of the invention is composed substantially entirely of thedesired immunogenic PRV polypeptide and/or other PRV polypeptidesdisplaying PRV antigenicity.

[0134] The immunogen can be prepared in vaccine dose form by well-knownprocedures. The vaccine can be administered intramuscularly,subcutaneously or intranasally. For parenteral administration, such asintramuscular injection, the immunogen may be combined with a suitablecarrier, for example, it may be administered in water, saline orbuffered vehicles with or without various adjuvants or immunomodulatingagents including aluminum hydroxide, aluminum phosphate, aluminumpotassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon,water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide,bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacteriumacnes), Bordetella pertussis, polyribonucleotides, sodium alginate,lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole,DEAE-dextran, blocked copolymers or other synthetic adjuvants. Suchadjuvants are available commercially from various sources, for example,Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Anothersuitable adjuvant is Freund's Incomplete Adjuvant (Difco Laboratories,Detroit, Mich.).

[0135] The proportion of immunogen and adjuvant can be varied over abroad range so long as both are present in effective amounts. Forexample, aluminum hydroxide can be present in an amount of about 0.5% ofthe vaccine mixture (Al₂O₃ basis). On a per dose basis, theconcentration of the immunogen can range from about 1.0 μg to about 100mg per pig. A preferable range is from about 100 μg to about 3.0 mg perpig. A suitable dose size is about 1-10 ml, preferably about 1.0 ml.Accordingly, a dose for intramuscular injection, for example, wouldcomprise 1 ml containing 1.0 mg of immunogen in admixture with 0.5%aluminum hydroxide. Comparable dose forms can also be prepared forparenteral administration to baby pigs, but the amount of immunogen perdose will be smaller, for example, about 0.25 to about 1.0 mg per dose.

[0136] For vaccination of sows, a two dose regimen can be used. Thefirst dose can be given from about several months to about 5 to 7 weeksprior to farrowing. The second dose of the vaccine then should beadministered some weeks after the first dose, for example, about 2 to 4weeks later, and vaccine can then be administered up to, but prior to,farrowing. Alternatively, the vaccine can be administered as a single 2ml dose, for example, at about 5 to 7 weeks prior to farrowing. However,a 2 dose regimen is considered preferable for the most effectiveimmunization of the baby pigs. Semi-annual revaccination is recommendedfor breeding animals. Boars may be revaccinated at any time. Also, sowscan be revaccinated before breeding. Piglets born to unvaccinated sowsmay be vaccinated at about 3-10 days, again at 4-6 months and yearly orpreferably semi-annually thereafter.

[0137] The vaccine may also be combined with other vaccines for otherdiseases to produce multivalent vaccines. It may also be combined withother medicaments, for example, antibiotics. A pharmaceuticallyeffective amount of the vaccine can be employed with a pharmaceuticallyacceptable carrier or diluent to vaccinate animals such as swine,cattle, sheep, goats, and other mammals.

[0138] Other vaccines may be prepared according to methods well known tothose skilled in the art as set forth, for example, in I. Tizard, AnIntroduction to Veterinary Immunology, 2nd ed. (1982), which isincorporated herein by reference.

[0139] As set forth above, commercial vaccine PRV's have been found tohave the gI and gp63 genes deleted. Therefore gI and gp63 polypeptidesproduced by the methods of this invention can be used as diagnosticagents to distinguish between animals vaccinated with these commercialvaccines and those infected with virulent virus.

[0140] To differentiate between infected and vaccinated animals, onecould employ, for example, an ELISA assay. gI or gp63 protein, produced,for example, in E. coli by recombinant DNA techniques (Rea, et al.,supra.), is added to the wells of suitable plastic plates and allowedsufficient time to absorb to the plastic (e.g., overnight, 20-25^(•)C.). The plates are washed and a blocking agent (e.g., BSA) is added toneutralize any unreacted sites on the plastic surface. A wash followsand then the pig serum is added to the wells. After about 1 hourincubation at 20-25^(•) C., the wells are washed and a proteinA-horseradish peroxidase conjugate is added to each well for an about 1hour incubation a 20-25^(•) C. Another wash follows and the enzymesubstrate (o-phenylenediamine) is added to the wells and the reaction isterminated with acid. Absorbency is measured at 492 nanometers toquantitate the amount of gI or gp63 antibody present in the serum. Lackof gI or gp63 indicates that an animal is not infected. By testing forother PRV antigens, one could establish whether or not a given animalwas vaccinated. CHART A                                 27                                  54ATG CTG CTC GCA GCG CTA TTC GCG GCG GTC CTC GCC CGG ACG ACG CTC GCT GCGMet Leu Leu Ala Ala Leu Leu Ala Ala Leu Val Ala Arg Thr Thr Leu Gly Ala                                 81                                 108GAC GTG GAC GCC GTG CCC GCG CCG ACC TTC CCC CCG CCC GCG TAC CCG TAC ACCAsp Val Asp Ala Val Pro Ala Pro Thr Phe Pro Pro Pro Ala Tyr Pro Tyr Thr                                135                                 162GAG TCG TGG CAG CTG ACG CTG ACG ACG GTC CCC TCG CCC TTC GTC GGC CCC GCGGlu Ser Trp Gln Leu Thr Leu Thr Thr Val Pro Ser Pro Phe Val Gly Pro Ala                                189                                 216GAC GTC TAC CAC ACG CGC CCG CTG GAG GAC CCG TGC GCG GTG GTG GCG CTG ATCAsp Val Tyr His Thr Arg Pro Leu Glu Asp Pro Gys Gly Val Val Ala Leu Ile                                243                                 270TCC GAC CCG CAG GTG GAC CGG CTG CTG AAC GAG GCG GTG GCC CAC CGG CGG CCCSer Asp Pro Gln Val Asp Arg Leu Leu Asn Glu Ala Val Ala His Arg Arg Pro                                297                                 324ACG TAC CGC GCC CAC GTG GCC TGG TAC CGC ATC GCG GAC GGG TGC GCA CAC CTGThr Tyr Arg Ala His Val Ala Trp Tyr Arg Ile Ala Asp Gly Cys Ala His Leu                                351                                 378CTG TAC TTT ATC GAG TAC GCC GAC TGC GAC CCC AGG CAG GTC TTT GGG CGC TGCLeu Tyr Phe Ile Glu Tyr Ala Asp Cys Asp Pro Arg Gln Val Phe Gly Arg Cys                                405                                 432CGG CGC CGC ACC ACG CCG ATG TGG TGG ACC CCG TCC GCG GAC TAC ATG TTC CCCArg Arg Arg Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe Pro                                459                                 486ACG GAG GAC GAG CTG GGG CTG CTC ATG GTG GCC CCG GGG CGG TTC AAC GAG GGCThr Glu Asp Glu Leu Gly Leu Leu Met Val Ala Pro Gly Arg Phe Asn Glu Gly                                513                                 540CAG TAC CGG CGC CTG GTC TCC GTC GAC GGC GTG AAC ATC CTC ACC GAC TTC ATGGln Tyr Arg Arg Leu Val Ser Val Asp Gly Val Asn Ile Leu Thr Asp Phe Met                                567                                 594GTG GCG CTC CCC GAG GGG CAA GAG TGC CCG TTC GCC GGC GTG GAC CAG CAC GGCVal Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala Arg Val Asp Gln His Arg                                621                                 648ACG TAC AAG TTC GGC GCG TGC TGG AGC CAC GAC AGC TTC AAG CGG GGC GTG GACThr Tyr Lys Phe Gly Ala Gys Trp Ser Asp Asp Ser Phe Lys Arg Gly Val Asp                                675                                 702GTG ATG CGA TTC CTG ACG CCG TTC TAC CAG CAG CCC CCG CAC CCG GAC GTG GTGVal Met Arg Phe Leu Thr Pro Phe Tyr Gln Gln Pro Pro His Arg Glu Val Val                                729                                 756AAC TAC TGG TAC CGC AAG AAC GGC CGG ACG CTC CCG CGG GCC CAC GCC GCC GCCAsn Tyr Trp Tyr Arg Lys Asn Gly Arg Thr Leu Pro Arg Ala His Ala Ala Ala                                783                                 810ACG CCG TAC GCC ATC GAC CCC GCG CGG CCC TCG GCG GGC TCG CCG AGG CCC CGCThr Pro Tyr Ala Ile Asp Pro Ala Arg Pro Ser Ala Gly Ser Pro Arg Pro Arg                                837                                 864CCC CGG CCC CGG CCC CGG CCC CGG CCG AAG CCC GAG CCC GCC CCG GCG ACG CCCPro Arg Pro Arg Pro Arg Pro Arg Pro Lys Pro Glu Pro Ala Pro Ala Thr Pro                                891                                 918GCG CCC CCC GAC CGC CTG CCC GAG CCG GCG ACG CGG GAC CAC GCC GCC GGG GGCAla Pro Pro Asp Arg Leu Pro Glu Pro Ala Thr Arg Asp His Ala Ala Gly Gly                                945                                 972CGC CCC ACG CCG CGA CCC CCG AGG CCC GAG ACG CCG CAC CGC CCC TTC GCC CCGArg Pro Thr Pro Arg Pro Pro Arg Pro Glu Thr Pro His Arg Pro The Ala Pro                                999                                1026CCG GCC GTC GTG CCC AGC GGG TGG CCG CAG CCC GCG GAG CCG TTC CAG CCG CGGPro Ala Val Val Pro Ser Gly Trp Pro Gln Pro Ala Glu Pro Phe Gln Pro Arg                               1053                                1080ACC CCC GCC GCG CCG GGC GTC TCG CGC CAC CGC TCG GTG ATC GTC GGC ACG GGCThr Pro Ala Ala Pro Gly Val Ser Arg His Arg Ser Val Ile Val Gly Thr Gly                               1107                                1134ACC GCG ATG GGC GCG CTC CTG GTG GGC GTG TGC CTC TAC ATC TTC TTC CGC CTGThr Ala Met Gly Ala Leu Leu Val Gly Val Cys Val Tyr Ile Phe Phe Arg Leu                               1161                                1188AGG GGG GCG AAG GGG TAT CGC CTC CTG GGC GGT CCC GCG GAC GCC GAC GAG CTAArg Gly Ala Lys Gly Tyr Arg Leu Leu Gly Gly Pro Ala Asp Ala Asp Glu Leu                               1215 AAA GCG CAG CCC GGT CCG TAG Lys AlaGln Pro Gly Pro

[0141] CHART B                                 27                                  54ATG ATG ATG GTG GCG GGC GAC GTG ACC CGG CTC CCC GCC GGG CTC CTC CTC GCCMet Met Met Val Ala Arg Asp Val Thr Arg Leu Pro Ala Gly Leu Leu Leu Ala                                 81                                 108GCC CTG ACC CTG GCC GCC CTG ACC CCG CGC GTC GGG GGC GTC CTC TTC AGG GGCAla Leu Thr Leu Ala Ala Leu Thr Pro Arg Val Gly Gly Val Leu Phe Arg Gly                                135                                 162GCC GGC GTC AGC GTG CAC GTC GCC GGG AGC GCC GTC CTC GTG CCC GGC GAC GCGAla Aly Val Ser Val His Val Ala Gly Ser Ala Val Leu Val Pro Gly Asp Ala                                189                                 216CCC AAC CTG ACG ATC GAC GGG ACG CTG CTG TTT CTG GAG GGG CCC TGC GCG AGCPro Asn Leu Thr Ile Asp Gly Thr Leu Leu Phe Leu Gly Gly Pro Ser Pro Ser                                243                                 270AAC TAC AGC GGG CGC GTG GAG CTG CTG CGC CTC GAC CCC AAG CGC GCC TGC TACAsn Tyr Ser Gly Arg Val Glu Leu Leu Arg Leu Asp Pro Lys Arg Ala Cys Tyr                                297                                 324ACG CGC GAG TAC GCC GCC GAG TAC GAC CTC TGC CCC CGC GTG CAC CAC GAG GCCThr Arg Glu Tyr Ala Ala Glu Tyr Asp Leu Cys Pro Arg Val His His Glu Ala                                351                                 378TTC CGC GGC TGT CTG CGC AAG CGC GAG CCG CTC GCC CGG GGC GCG TCC GCC GCGPhe Arg Gly Cys Leu Arg Lys Arg Glu Pro Leu Ala Arg Arg Ala Ser Ala Ala                                405                                 432GTG GAG GCG CGC CGG CTG CTG TTC GTC TCG CGC CCG GCC CCG CCG CAC GCG GGGVal Glu Ala Arg Arg Leu Leu Phe Val Ser Arg Pro Ala Pro Pro Asp Ala Gly                                459                                 486TCG TAC GTG CTG CGG CTC CGC GTG AAC GGG ACC ACG GAC CTC TTT GTG CTG ACGSer Tyr Val Leu Arg Val Arg Val Asn Gly Thr Thr Asp Leu Phe Val Leu Thr                                513                                 540GCC CTG GTG CCG CCC AGG GGG CGC CCC CAC CAC CCC ACG CCG TGG TCC GCG GACAla Leu Val Pro Pro Arg Gly Arg Pro His His Pro Thr Pro Ser Ser Ala Asp                                567                                 594GAG TGC CGG CCT GTC GTC GGA TCG TGG CAC GAC AGC CTG CGC GTC GTG GAC CCCGlu Cys Arg Pro Val Val Gly Ser Trp His Asp Ser Leu Arg Val Val Asp Pro                                621                                 648GCC GAG GAC GCC GTG TTC ACC ACG CCG CCC CCG ATC GAG CCA GAG CCG CCG ACGAla Glu Asp Ala Val Phe Thr Thr Pro Pro Pro Ile Glu Pro Glu Pro Pro Thr                                675                                 702ACC CCC GCG CCC CCC CGG GGG ACC GGC GCC ACC CCC GAG CCC CGC TCC GAC GAAThr Pro Ala Pro Pro Arg Gly Thr Gly Ala Thr Pro Glu Pro Arg Ser Asp Glu                                729                                 756GAG GAG GAG GAC CAG GAC GGG GCG ACG ACG GCG ATC ACC CCG CTG CCC GGG ACCGlu Glu Glu Asp Glu Glu Gly Ala Thr Thr Ala Met Thr Pro Val Pro Gly Thr                                783                                 810CTG GAC GCG AAC GGC ACG ATG GTG CTG AAC GCC AGC GTC GTG TCG CGC GTC CTGLeu Asp Ala Asn Gly Thr Met Val Leu Asn Ala Ser Val Val Ser Arg Val Leu                                837                                 864CTC GCC GCC GCC AAC GCC ACG GCG GGC GCC CGG GGC CCC GGG AAG ATA GCC ATGLeu Ala Ala Ala Asn Ala Thr Ala Gly Ala Arg Gly Pro Gly Lys Ile Ala Met                                891                                 918GTG CTG GGG CCC ACG ATC GTC GTC CTC CTG ATC TTC TTG GGC GGG GTC GCC TGCVal Leu Gly Pro Thr Ile Val Val Leu Leu Ile Phe Leu Gly Gly Val Ala Cys                                945                                 972GCG GCC CGG CGC TGC GCG CGC CGA ATC GCA TCT ACC GGC CGC GAC CCG GGC GCGAla Ala Arg Arg Cys Ala Arg Gly Ile Ala Ser Thr Gly Arg Asp Pro Gly Ala                                999                                1026GCC CGG CGG TCC ACG CGC CGC CCC CGC CGC GCC CGC CCC CCA ACC CCG TCG CCGAla Arg Arg Ser Thr Arg Arg Pro Arg Gly Ala Arg Pro Pro Thr Pro Ser Pro                               1053 GGG CGC CCG TCC CCC AGC CCA AGA TGAGly Arg Pro Ser Pro Ser Pro Arg

[0142] CHART C                                 27                                  54ATG CGG CCC TTT CTG CTG CGC GCC GCG CAG CTC CTG GCG CTG CTG GCC CTG GCGMet Arg Pro Phe Leu Leu Arg Ala Ala Gln Leu Leu Ala Leu Leu Ala Leu Ala                                 81                                 108CTC TCC ACC GAG GCC CCG AGC CTC TCC GCC GAG ACG ACC CCG GGC CCC GTC ACCLeu Ser Thr Glu Ala Pro Ser Leu Ser Ala Glu Thr Thr Pro Gly Pro Val Thr                                135                                 162GAG GTC CCG AGT CCC TCG GCC GAG GTC TGG GAC CTC TCC ACC GAG GCC GGC GACGlu Val Pro Ser Pro Ser Ala Glu Val Trp Asp Leu Ser Thr Glu Ala Gly Asp                                189                                 216GAT GAC CTC GAC GGC GAC CTC AAC GGC GAC GAC CGC CGC GCG GGC TTC GGC TCGAsp Asp Leu Asp Gly Asp Leu Asn Gly Asp Asp Arg Arg Ala Gly Phe Gly Ser                                243                                 270GCC CTC GCC TCC CTG AGG GAG GCA CCC CCG GCC CAT CTG GTG AAC GTG TCC GAGAla Leu Ala Ser Leu Arg Glu Ala Pro Pro Ala His Leu Val Asn Val Ser Glu                                297                                 324GGC GCC AAC TTC ACC CTC GAC GCG CGC GGC GAC GGC GCC GTG GTG GCC GGG ATCGly Ala Asn Phe Thr Leu Asp Ala Arg Gly Asp Gly Ala Val Val Ala Gly Ile                                351                                 378TGG ACG TTC CTG CCC GTC GGC GGC TGC GAC GCC GTG GCG GTG ACC ATG GTG TGCTrp Thr Phe Leu Pro Val Arg Gly Cys Asp Ala Val Ala Val Thr Met Val Cys                                405                                 432TTC GAG ACC GCC TGC CAC CCG GAC CTG GTG CTG GGC CGC GCC TGC GTC CCC GAGPhe Glu Thr Ala Cys His Pro Asp Leu Val Leu Gly Arg Ala Cys Val Pro Glu                                459                                 486GCC CCG GAG CGG GGC ATC GGC GAC TAC CTG CCG CCC GAG GTG CCG CGG CTC CAGAla Pro Glu Arg Gly Ile Gly Asp Tyr Leu Pro Pro Glu Val Pro Arg Leu Gln                                513                                 540CGC GAG CCG CCC ATC GTC ACC CCG GAG CCG TGG TCG CCC CAC CTG ACC TGC CGGArg Glu Pro Pro Ile Val Thr Pro Glu Arg Trp Ser Pro His Leu Thr Val Arg                                567                                 594CGG GCC ACG CCC AAC GAC ACG GGC CTC TAC ACG CTG CAC GAC GCC TCG GCG CCGArg Ala Thr Pro Asn Asp Thr Gly Leu Tyr Thr Leu His Asp Ala Ser Gly Pro                                621                                 648CGG GCC GTG TTC TTT GTG GCG GTG GGC GAC CGG CCG CCC GCG CCG CTG GCC CCGArg Ala Val Phe Phe Val Ala Val Gly Asp Arg Pro Pro Ala Pro Leu Ala Pro                                675                                 702GTG GGC CCC GCG CGC CAC GAG CCC CGC TTC CAC GCG CTC GGC TTC CAC TCG GAGVal Gly Pro Ala Arg His Glu Pro Arg Phe His Ala Leu Gly Phe His Ser Gln                                729                                 756CTC TTC TCG CCC GGG GAC ACG TTC GAC CTG ATG CCG CGC GTG GTC TCG GAC ATGLeu Phe Ser Pro Gly Asp Thr Phe Asp Leu Met Pro Arg Val Val Ser Asp Met                                783                                 810GGC GAC TCG CGC GAG AAC TTC ACC GCC ACG CTG GAC TGG TAC TAC GCG CGC GCGGly Asp Ser Arg Glu Asn Phe Thr Ala Thr Leu Asp Trp Tyr Tyr Ala Arg Ala                                837                                 864CCC CCG CGG TGC CTG CTG TAC TAC GTG TAC GAG CCC TGC ATC TAC CAC CCG CGCPro Pro Arg Cys Leu Leu Tyr Tyr Val Tyr Glu Pro Cys Ile Tyr His Pro Arg                                891                                 918GCG CCC GAG TGC CTG CGC CCG GTG GAC CCG GCG TCC AGC TTC ACC TCG CCG GCGAla Pro Glu Cys Leu Arg Pro Val Asp Pro Ala Cys Ser Phe Thr Ser Pro Ala                                945                                 972CGC GCG GCG CTG GTG GCG CGC CGC GCG TAC GCC TCG TGC ACC CCG CTG CTC GGGArg Ala Ala Leu Val Ala Arg Arg Ala Tyr Ala Ser Cys Ser Pro Leu Leu Gly                                999                                1026GAC CGG TGG CTG ACC GCC TGC CCC TTC GAC GCC TTC GGC GAG GAG GTG CAC ACGAsp Arg Trp Leu Thr Ala Gys Pro Phe Asp Ala Phe Gly Glu Glu Val His Thr                               1053                                1080AAC GCC ACC GCG GAC GAG TCG GGG CTG TAC GTG CTC GTG ATG ACC CAC AAC GGCAsn Ala Thr Ala Asp Glu Ser Gly Leu Tyr Val Leu Val Met Thr His Asn Gly                               1107                                1134CAC GTC GCC ACC TGG GAC TAC ACG CTC GTC GCC ACC GCG GCC GAG TAC GTC ACGHis Val Ala Thr Trp Asp Tyr Thr Leu Val Ala Thr Ala Ala Glu Tyr Val Thr                               1161                                1188GTC ATC AAG GAG CTG ACG GCC CCG GCC CGG GCC CCG GGC ACC CCG TGG GGC CCCVal Ile Lys Glu Leu Thr Ala Pro Ala Arg Ala Pro Gly Thr Pro Trp Gly Pro                               1215                                1242GGC GGC GGC GAC GAC GCG ATC TAC GTG GAC GGC GTC ACG ACG CCG GCG CCG CCCGly Gly Gly Asp Asp Ala Ile Tyr Val Asp Gly Val Thr Thr Pro Ala Pro Pro                               1269                                1296GCG CGC CCG TGG AAC CCG TAC GGC CGG ACG ACG CCC GGG GGG CTG TTT GTG CTGAla Arg Pro Trp Asn Pro Tyr Gly Arg Thr Thr Pro Gly Arg Leu Phe Val Leu                               1323                                1350GCG CTG GGC TCC TTC GTG ATG ACG TGC GTC GTC GGG GGG GCC CTC TGG CTC TGCAla Leu Gly Ser Phe Val Met Thr Cys Val Val Gly Gly Ala Val Trp Leu Cys                               1377                                1404GTG CTG TGC TCC CGC CGC CGG GCG GCC TCG CGG CCG TTC CGG GTG CCG ACG CGGVal Leu Gys Ser Arg Arg Arg Ala Ala Ser Arg Pro Phe Arg Val Pro Thr Arg                               1431                                1458GCG GGG ACG CGC ATG CTC TCG CCG GTG TAC ACC AGC CTG CCC ACG CAC GAG GACAla Gly Thr Arg Her Leu Ser Pro Val Tyr Thr Ser Leu Pro Thr His Glu Asp                               1485                                1512TAC TAC GAC GGC GAC GAC GAC GAC GAG GAG GCG GGC GAC GCC CGC CGG CGG CCCTyr Tyr Asp Gly Asp Asp Asp Asp Glu Glu Ala Gly Asp Ala Arg Arg Arg Pro                               1539                                1566TCC TCC CCC GGC GGG GAC AGC GGC TAC GAG GGG CCG TAC GTG AGC CTG GAC GCCSer Ser Pro Gly Gly Asp Ser Gly Tyr Glu Gly Pro Tyr Val Ser Leu Asp Ala                               1593                                1620GAG GAC GAG TTC AGC AGC GAC GAG GAC GAC CGG CTG TAC GTG CGC CCC GAG GAGGlu Asp Glu Phe Ser Ser Asp Glu Asp Asp Gly Leu Tyr Val Arg Pro Glu Glu                               1647                                1674GCG CCC CGC TCC GGC TTC GAC GTC TGG TTC CGC GAT CCG GAG AAA CCG GAA GTGAla Pro Arg Ser Gly Phe Asp Val Trp Phe Arg Asp Pro Glu Lys Pro Glu Val                               1701                                1728ACG AAT GGG CCC AAC TAT GGC GTG ACC GCC AGC CGC CTG TTG AAT GCC CGC CCCThr Asn Gly Pro Asn Tyr Gly Val Thr Ala Ser Arg Leu Leu Asn Ala Arg Pro                               1755 GCT TAA Ala

[0143]

CHART Q Construction of Plasmid pDIEGIPA (a) Plasmid pDGI is cut withBamHI to produce fragment 6.

(b) Fragment 7 (760 bp) containing the human cytomegalovirus (Towme)immediate early promoter is isolated.

(c) Fragments 6 and 7 are ligated to produce plasmid pDIEGIdhfr.

(d) Plasmid pSVCOW7 is cut with PvuII and EcoRI to produce fragment 8.

Fragment 8

(e) Fragment 8 is cloned in pUC9 to produce plasmid pCOWT1.

(f) pCOWT1 is cut with SalI, treated with T4 DNA polymerase, and EcoRIlinkers are ligated on followed by digestion with EcoRI to producefragment = 9 (0.6 kb).

(g) Fragment 9 is cloned into the EcoRI site of pUC19 to produce plasmidpCOWT1E.

(h) pCOWT1E is digested with EcoRI to produce fragment 10 (600 bp).

(i) Plasmid pDIEGIdhfr is digested with EcoRI and ligated with fragment10 containing the bGH polyadenylation signal to produce plasmidpDIEGIPA.

[0144] CHART R Construction of pGP63dhfr (a) BamHI 7 is digested withBstEII. treated with T4 DNA polymerase. and then cut with KpnI to yieldfragment 1 (1.9 kb).

(b) Fragment 1 is then cloned between the KpnI and SmaI sites of plasmidpUC19 to yield plasmid pPR28-1BK.

(c) Plasmid pPR28-1BK is cut with DraI and MaeIII to yield fragment 2(1.1 kb).

(d) Plasmid pSV2dhfr is cut with EcoRI, treated with T4 DNA poly-merase, and then cut with BamHI to obtain fragment 3 (5.0 kb).

(e) Two oligonucleotides are synthesized to produce fragment 4.

(f) Fragments 2, 3, and 4 are then ligated to produce plasmid pGP63dhfr

[0145] CHART S (a) pGP63dhfr is digested with BamHI and treated with BAPto produce fragment 5.

(b) Fragment 6 (760 bp) containing the human cytomegalovirus (Towne)immediate early promoter is isolated.

(c) Fragments 5 and 6 are ligated to produce plasmid pIEGP63dhfr.

1. A recombinant DNA molecule comprising a DNA sequence coding for apolypeptide displaying pseudorabies virus (PRV) glycoprotein gp50 orgp63, said DNA sequence being operatively linked to an expressioncontrol sequence.
 2. A recombinant DNA molecule of claim 1, wherein theDNA sequence coding for the polypeptide is selected from the groupconsisting of the sequence coding for gp50, which is ATG CTG CTC GCA GCGCTA TTG GCG GCG GTG GTC GCC CGG ACG ACG GTG GGT GCG GAG GTG GAC GCC GTGCCC GCG CCG ACC TTC CCC CCG CCC GCG TAC CCG TAC ACC GAG TCG TGG CAG CTGACG CTG ACG ACG GTC CCC TCC CCC TTC GTC GGC CCC GCG GAC GTC TAC CAC ACGCGC CCG CTG GAG GAC CCG TGC GCG GTG GTG GCG CTG ATC TCC GAC CCG CAG GTGGAC CGG CTG CTG AAC GAG GCG GTG GCC CAC CGG CGG CCC ACG TAC CGC GCC CACGTG GCC TGG TAC CGC ATC GCG GAC GGG TGC GCA CAC CTG CTG TAC TTT ATC GAGTAC GCC GAC TGC GAC CCC AGG CAG CTC TTT GGG CGC TGC CGG CGC CGC ACC ACGCCG ATG TGG TGG ACC CCG TCC GCG GAC TAC ATG TTC CCC ACG GAG GAC GAG CTGGGG CTG CTC ATG GTG GCC CCG GGG CGG TTC AAC GAG GGC CAG TAC CGG GCG CTGGTG TCC GTC GAC GGC GTG AAC ATC CTC ACC GAC TTC ATG GTG GCG CTC CCC GAGGGG CAA GAG TGC CCG TTC GCC CGC GTG GAC CAG CAC CGC ACG TAC AAG TTC GGCGCG TGC TGG AGC GAC GAC AGC TTC AAG GGG GGC GTG GAC GTG ATG CGA TTC CTGACG CCG TTC TAC CAG CAG CCC CCG CAC CGG GAG GTG GTG AAC TAC TGG TAC CGCAAG AAC GGC CGG ACG CTC CCG CGG GCC CAC GCC GCC GCC ACG CCG TAC GCC ATCGAC CCC GCG CGG CCC TCG GCG GGC TCG CCG AGG CCC CGG CCC CGG CCC CGG CCCCGG CCC CGG CCG AAG CCC GAG CCC GCC CCG GCG ACG CCC GCG CCC CCC GAC CGCCTG CCC GAG CCG GCG ACG CGG GAC CAC GCC GCC GGG GGC CGC CCC ACG CCG CGACCC CCG AGG CCC GAG ACG CCG CAC CGC CCC TTC GCC CCG CCG GCC GTC GTG CCCAGC GGG TGG CCG CAG CCC GCG GAG CCG TTC CAG CCG CGG ACC CCC GCC GCG CCGGGC GTC TCG CGC CAC CGC TCG GTG ATC GTC GGC ACG GGC ACC GCG ATG GGC GCGCTC CTG GTG GGC GTG TGC GTC TAC ATC TTC TTC CGC CTG AGG GGG GCG AAG GGGTAT CGC CTC CTG GGC GGT CCC GCG GAC GCC GAC GAG CTA AAA GCG CAG CCC GGTCCG TAG and the sequence coding for gp63, which is ATG ATG ATG GTG GCGCGC GAC GTG ACC CGG CTC CCC GCG GGG CTC CTC CTC GCC GCC CTG ACC CTG GCCGCC CTG ACC CCG CGC GTC GGG GGC GTC CTC TTC AGG GGC GCC GGC GTC AGC GTGCAC GTC GCC GGG AGC GCC GTC CTC GTG CCC GGC GAC GCG CCC AAC CTG ACG ATCGAC GGG ACG CTG CTG TTT CTG GAG GGG CCC TCG CCG AGC AAC TAC AGC GGG CGCGTG GAG CTG CTG CGC CTC GAC CCC AAG CGC GCC TGC TAC ACG CGC GAG TAC GCCGCC GAG TAC GAC CTC TGC CCC CGC GTG CAC CAC GAG GCC TTC CGC GGC TGT CTGCGC AAG CGC GAG CCG CTC GCC CGG CGC GCG TCC GCC GCG GTG GAG GCG CGC CGGCTG CTG TTC GTC TCG GGC CCG GCC CCG CCG GAC GCG GGG TCG TAC GTG CTG CGGGTC CGC GTG AAC GGG ACC ACG GAC CTC TTT GTG CTG ACG GCC CTG GTG CCG CCCAGG GGG CGC CCC CAC CAC CCC ACG CCG TCG TCC GCG GAC GAG TGC CGG CCT GTCCTC GGA TCG TGG CAC GAC AGC CTC CGC GTC GTG GAC CCC GCC GAG GAC GCC GTGTTC ACC ACG CCG CCC CCG ATC GAG CCA GAG CCG CCG ACG ACC CCC GCG CCC CCCCGG GGG ACC GGC GCC ACC CCC GAG CCC CGC TCC GAC GAA GAG GAG GAG GAC GAGGAG GGG GCG ACG ACG GCG ATG ACC CCC GTG CCC GGG ACC CTG GAC GCG AAC GGCACG ATG GTG CTG AAC GCC AGC GTC GTG TCG CGC GTC CTC CTC GCC GCC GCC AACGCC ACG GCG GGC GCC CGG GGC CCC GGG AAG ATA GCC ATG GTG CTG GGG CCC ACGATC GTC GTC CTC CTG ATC TTC TTG GCC GGG GTC GCC TGC GCG GCC CGG CGC TGCGCG CGC GGA ATC GCA TCT ACC GGC CGC GAC CCG GGC GCG GCC CGG CGG TCC ACGCGC CGC CCC CGC GGC GCC CGC CCC CCA ACC CCG TCG CCC GGG CGC CCG TCC CCCAGC CCA AGA TGA

and fragments thereof encoding polypeptides displaying PRV antigenicity.3. A host cell transformed with a recombinant DNA molecule of claim 1.4. A host cell of claim 3 which is of bacterial, fungal, plant or animalorigin.
 5. A host cell of claim 4 which is E. coli.
 6. A host cell ofclaim 4 which is a yeast cell.
 7. A host cell of claim 4 which is aChinese hamster ovary (CHO) cell.
 8. An essentially pure polypeptideselected from the group consisting of: the gp50 polypeptide, which isMet Leu Leu Ala Ala Leu Leu Ala Ala Leu Val Ala Arg Thr Thr Leu Gly AlaAsp Val Asp Ala Val Pro Ala Pro Thr Phe Pro Pro Pro Ala Tyr Pro Tyr ThrGlu Ser Trp Gln Leu Thr Leu Thr Thr Val Pro Ser Pro Phe Val Gly Pro AlaAsp Val Tyr His Thr Arg Pro Leu Gln Asp Pro Cys Gly Val Val Ala Leu IleSer Asp Pro Gln Val Asp Arg Leu Leu Asn Glu Ala Val Ala His Arg Arg ProThr Tyr Arg Ala His Val Ala Trp Tyr Arg Ile Ala Asp Gly Cys Ala His LeuLeu Tyr Phe Ile Gln Tyr Ala Asp Cys Asp Pro Arg Gln Val Phe Gly Arg CysArg Arg Arg Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe ProThr Gln Asp Gln Leu Gly Leu Leu Met Val Ala Pro Gly Arg Phe Asn Gln GlyGln Tyr Arg Arg Leu Val Ser Val Asp Gly Val Asn Ile Leu Thr Asp Phe MetVal Ala Leu Pro Gln Gly Gln Gln Cys Pro Phe Ala Arg Val Asp Gln His ArgThr Tyr Lys Phe Gly Ala Cys Trp Ser Asp Asp Ser Phe Lys Arg Gly Val AspVal Met Arg Phe Leu Thr Pro Phe Tyr Gln Gln Pro Pro His Arg Glu Val ValAsn Tyr Trp Tyr Arg Leu Asn Gly Arg Thr Leu Pro Arg Ala His Ala Ala AlaThr Pro Tyr Ala Ile Asp Pro Ala Arg Pro Ser Ala Gly Ser Pro Arg Pro ArgPro Arg Pro Arg Pro Arg Pro Arg Pro Leu Pro Gln Pro Ala Pro Ala Thr ProAla Pro Pro Asp Arg Leu Pro Gln Pro Ala Thr Arg Asp His Ala Ala Gly GlyArg Pro Thr Pro Arg Pro Pro Arg Pro Gln Thr Pro His Arg Pro Phe Ala ProPro Ala Val Val Pro Ser Gly Trp Pro Gln Pro Ala Glu Pro Phe Gln Pro ArgThr Pro Ala Ala Pro Gly Val Ser Arg His Arg Ser Val Ile Val Gly Thr GlyThr Ala Met Gly Ala Leu Leu Val Gly Val Cys Val Tyr Ile Phe Phe Arg LeuArg Gly Ala Leu Gly Tyr Arg Leu Leu Gly Gly Pro Ala Asp Ala Asp Gln LegLys Ala Gln Pro Gly Pro and to gp63 polypeptide, which is Met Met MetVal Ala Arg Asp Val Thr Arg Leu Pro Ala Gly Leu Leu Leu Ala Ala Leu ThrLeu Ala Ala Leu Thr Pro Arg Val Gly Gly Val Leu Phe Arg Gly Ala Aly ValSer Val His Val Ala Gly Ser Ala Val Leu Val Pro Gly Asp Ala Pro Asn LeuThr Ile Asp Gly Thr Leu Leu Phe Leu Glu Gly Pro Ser Pro Ser Asn Tyr SerGly Arg Val Glu Leu Leu Arg Leu Asp Pro Lys Arg Ala Cys Tyr Thr Arg GluTyr Ala Ala Glu Tyr Asp Leu Cys Pro Arg Val His His Glu Ala Phe Arg GlyCys Leu Arg Lys Arg Glu Pro Leu Ala Arg Arg Ala Ser Ala Ala Val Glu AlaArg Arg Leu Leu Phe Val Ser Arg Pro Ala Pro Pro Asp Ala Gly Ser Tyr ValLeu Arg Val Arg Val Asn Gly Thr Thr Asp Leu Phe Val Leu Thr Ala Leu ValPro Pro Arg Gly Arg Pro His His Pro Thr Pro Ser Ser Ala Asp Glu Cys ArgPro Val Val Gly Ser Trp His Asp Ser Leu Arg Val Val Asp Pro Ala Glu AspAla Val Phe Thr Thr Pro Pro Pro Ile Glu Pro Glu Pro Pro Thr Thr Pro AlaPro Pro Arg Gly Thr Gly Ala Thr Pro Glu Pro Arg Ser Asp Glu Glu Glu GluAsp Glu Glu Gly Ala Thr Thr Ala Met Thr Pro Val Pro Gly Thr Leu Asp AlaAsn Gly Thr Met Val Leu Asn Ala Ser Val Val Ser Arg Val Leu Leu Ala AlaAla Asn Ala Thr Ala Gly Ala Arg Gly Pro Gly Lys Ile Ala Met Val Leu GlyPro Thr Ile Val Val Leu Leu Ile Phe Leu Gly Gly Val Ala Cys Ala Ala ArgArg Cys Ala Arg Gly Ile Ala Ser Thr Gly Arg Asp Pro Gly Ala Ala Arg ArgSer Thr Arg Arg Pro Arg Gly Ala Arg Pro Pro Thr Pro

Ser Pro Gly Arg Pro Ser Pro Ser Pro Arg and fragments thereof displayingpseudorabies antigencity.
 9. A vaccine comprising a polypeptidedisplaying pseudorabies virus gp50 antigenicity.
 10. A vaccine accordingto claim 9, wherein the polypeptide is gp50 and is of the followingsequence: Met Leu Leu Ala Ala Leu Leu Ala Ala Leu Val Ala Arg Thr ThrLeu Gly Ala Asp Val Asp Ala Val Pro Ala Pro Thr Phe Pro Pro Pro Ala TyrPro Tyr Thr Glu Ser Trp Gln Leu Thr Leu Thr Thr Val Pro Ser Pro Phe ValGly Pro Ala Asp Val Tyr His Thr Arg Pro Leu Glu Asp Pro Cys Gly Val ValAla Leu Ile Ser Asp Pro Gln Val Asp Arg Leu Leu Asn Glu Ala Val Ala HisArg Arg Pro Thr Tyr Arg Ala His Val Ala Trp Tyr Arg Ile Ala Asp Gly CysAla His Leu Leu Tyr Phe Ile Glu Tyr Ala Asp Cys Asp Pro Arg Gln Val PheGly Arg Cys Arg Arg Arg Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp TyrMet Phe Pro Thr Glu Asp Glu Leu Gly Leu Leu Met Val Ala Pro Gly Arg PheAsn Glu Gly Gln Tyr Arg Arg Leu Val Ser Val Asp Gly Val Asn Lie Leu ThrAsp Phe Met Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala Arg Val AspGln His Arg Thr Tyr Lys Phe Gly Ala Cys Trp Ser Asp Asp Ser Phe Lys ArgGly Val Asp Val Met Arg Phe Leu Thr Pro Phe Tyr Gln Gln Pro Pro His ArgGlu Val Val Asn Tyr Trp Tyr Arg Lys Asn Gly Arg Thr Leu Pro Arg Ala HisAla Ala Ala Thr Pro Tyr Ala Ile Asp Pro Ala Arg Pro Ser Ala Gly Ser ProArg Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro Lys Pro Glu Pro Ala ProAla Thr Pro Ala Pro Pro Asp Arg Leu Pro Glu Pro Ala Thr Arg Asp His AlaAla Gly Gly Arg Pro Thr Pro Arg Pro Pro Arg Pro Glu Thr Pro His Arg ProPhe Ala Pro Pro Ala Val Val Pro Ser Gly Trp Pro Gln Pro Ala Glu Pro PheGln Pro Arg Thr Pro Ala Ala Pro Gly Val Ser Arg His Arg Ser Val Ile ValGly Thr Gly Thr Ala Met Gly Ala Leu Leu Val Gly Val Cys Val Tyr Ile PhePhe Arg Leu Arg Gly Ala Lys Gly Tyr Arg Leu Leu Gly Gly Pro Ala

Asp Ala Asp Glu Leu Lys Ala Gln Pro Gly Pro and fragments thereofdisplaying PRV antigencity.
 11. A method of protecting an animalsusceptible to PRV infection from said infection, comprisingadministering a vaccine of claim 9 to the animal.
 12. The methodaccording to claim 11, wherein said animal is a swine.
 13. The methodfor producing a polypeptide displaying PRV gp50 or gp63 antigenicity,comprising: (a) preparing a recombinant DNA molecule, said moleculecomprising a DNA sequence coding for a polypeptide displaying PRV gp50or gp63 antigenicity, said DNA sequence having operatively linkedthereto an expression control sequence; (b) transforming an appropriatehost cell with said recombinant DNA molecule; (c) culturing said hostcell; (d) and collecting said polypeptide.
 14. A method according toclaim 13, wherein the DNA sequence is selected from the group consistingof the gp50 sequence, which is ATG CTG CTC GCA GCG CTA TTG GCG GCG CTGGTG GCC CGG ACG ACG CTC GGT GCG GAC GTG GAC GCC GTG CCC GCG CCG ACC TTCCCC CCG CCC GCG TAC CCG TAC ACC GAG TCG TGG CAG CTG ACG CTG ACG ACG GTCCCC TCC CCC TTC GTC GGC CCC GCG GAC GTC TAC CAC ACG CGC CCG CTG GAG GACCCG TGC GCG GTG GTG GCG CTG ATC TCC GAC CCG CAG GTG GAC CGG CTG CTG AACGAG GCG GTG GCC CAC CGG CGG CCC ACG TAC CGC GCC CAC GTG GCC TGG TAC CGCATC GCG GAC GGG TGC GCA CAC CTG CTG TAC TTT ATC GAG TAC GCC GAC TGC GACCCC AGG CAG GTC TTT GGG CGC TGC CGG CGC CGC ACC ACG CCG ATG TGG TGG ACCCCG TCC GCG GAC TAC ATG TTC CCC ACG GAG GAC GAG CTG GGG CTG CTC ATG GTGGCC CCG GGG CGG TTC AAC GAG GGC CAG TAC CGG CGC CTG GTG TCC GTC GAC GGCGTG AAC ATC CTC ACC GAC TTC ATG GTG GCG CTC CCC GAG GGG CAA GAG TGC CCGTTC GCC CGC GTG GAC CAG CAC CGC ACG TAC AAG TTC GGC GCG TGC TGG AGC GACGAC AGC TTC AAG CGG GGC GTG GAC GTG ATG CGA TTC CTG ACG CCG TTC TAC CAGCAG CCC CCG CAC CGG GAG GTG GTG AAC TAC TGG TAC CGC AAG AAC GGC CGG ACGCTC CCG CGG GCC CAC GCC GCC GCC ACG CCG TAC GCC ATC GAC CCC GCG CGG CCCTCG GCG GGC TCG CCG AGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCG AAGCCC GAG CCC GCC CCG GCG ACG CCC GCG CCC CCC GAC CGC CTG CCC GAG CCG GCGACG CGG GAC CAC GCC GCC GGG GGC CGC CCC ACG CCG GGA CCC CCG AGG CCC GAGACG CCG CAC CGC CCC TTC GCC CCG CCG GCC GTC GTG CCC AGC GGG TGG CCG CAGCCC GCG GAG CCG TTC CAG CCG CGG ACC CCC GCC GCG CCG GGC GTC TCG CGC CACCGC TCG GTG ATC GTC GGC ACG GGC ACC GCG ATG GGC GCG CTC CTG GTG GGC GTGTGC GTC TAC ATC TTC TTC CGC CTG AGG GGG GCG AAG GGG TAT CGC CTC CTG GGCGGT CCC GCG GAC GCC GAC GAG CTA AAA GCG CAG CCC GGT CCG TAG and the gp63sequence, which is ATG ATG ATG GTG GCG CGC GAC GTG ACC CGG CTC CCC GCGGGG CTC CTC CTC GCC GCC CTG ACC CTG GCC GCC CTG ACC CCG CGC TGC GGG GGCGTC CTC TTC AGG GGC GCC GGC GTC AGC GTG CAC GTC GCC GGG AGC GCC GTC CTCGTG CCC GGC GAC GCG CCC AAC CTG ACG ATC GAC GGG ACG CTG CTG TTT CTG GAGGGG CCC TCG CCG AGC AAC TAC AGC GGG CGC GTG GAG CTG CTG CGC CTC GAC CCCAAG CGC GCC TGC TAC ACG CGC GAG TAC GCC GCC GAG TAC GAC CTC TGC CCC CGCGTG CAC CAC GAG GCC TTC CGC GGC TGT CTG CGC AAG CGC GAG CCG CTC GCC CGGCGC GCG TCC GCC GCG GTG GAG GCG CGC CGG CTG CTG TTC GTC TCG CGC CCG GCCCCG CCG GAC GCG GGG TCG TAC GTG CTG CGG GTC CGC GTG AAC GGG ACC ACG GACCTC TTT GTG CTG ACG GCC CTG GTG CCG CCC AGG GGG CGC CCC CAC CAC CCC ACGCCG TCG TCC GCG GAC GAG TGC CGG CCT GTC GTC GGA TCG TGG CAC GAC AGC CTGCGC GTC GTG GAC CCC GCC GAG GAC GCC GTG TTC ACC ACG CCG CCC CCG ATC GAGCCA GAG CCG CCG ACG ACC CCC GCG CCC CCC CGG GGG ACC GGC GCC ACC CCC GAGCCC CGC TCC GAC GAA GAG GAG GAG GAC GAG GAG GGG GCG ACG ACG GCG ATG ACCCCG GTG CCC GGG ACC CTG GAC GCG AAC GGC ACG ATG GTG CTG AAC GCC AGC GTCGTG TCG CGC GTC CTG CTC GCC GCC GCC AAC GCC ACG GCG GGC GCC CGG GGC CCCGGG AAG ATA GCC ATG GTG CTG GGG CCC ACG ATC GTC GTC CTC CTG ATC TTC TTGGGC GGG GTC GCC TGC GCG GCC CGG CGC TGC GCG CGC GGA ATC GCA TCT ACC GGCCGC GAC CCG GGC GCG GCC CGG CGG TCC ACG CGC CGC CCC CGC GGC GCC CGC CCCCCA ACC CCG TCG CCG GGG CGC CCG TCC CCC AGC CCA AGA TGA

and fragments thereof that encodes a polypeptide displaying pseudorabiesvirus antigenicity.
 15. A method according to claim 13, wherein the hostcell is selected from the group consisting of bacteria, fungi, plantcells and animal cells.
 16. A method-according to claim 13, wherein thehost cell is E. coli.
 17. A method according to claim 13, wherein thehost cell is yeast.
 18. A method according to claim 13, wherein the hostcell is CHO.
 19. A method for distinguishing between animals vaccinatedagainst PRV and those infected with PRV, comprising vaccinatingsusceptible animals with a PRV lacking glycoprotein gI or gp63 and thenserologically distinguishing between such vaccinated animals and thoseinfected with PRV without sacrificing the animal.
 20. A merchantile kituseful for performing the method of claim 19, comprising multiplecontainers wherein one of said containers has therein a polypeptidedisplaying PRV glycoprotein gI or gp63 antigenicity.